Antibodies directed to HER-3 and uses thereof

ABSTRACT

The present invention relates to binding proteins that bind to HER-3 and polynucleotides encoding the same. Expression vectors and host cells comprising the same for the production of the binding protein of the invention are also provided. In addition, the invention provides compositions and methods for diagnosing and treating diseases associated with HER-3 mediated signal transduction and/or its ligand heregulin.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/755,103 filed Dec. 30, 2005, incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to binding proteins including antibodiesand binding fragments thereof that bind to HER-3 and polynucleotidesencoding the same. Expression vectors and host cells comprising the samefor the production of the binding protein of the invention are alsoprovided. In addition, the invention provides compositions and methodsfor diagnosing and treating diseases associated with HER-3 mediatedsignal transduction and/or its ligand heregulin.

2. Background of the Technology

The human epidermal growth factor receptor 3 (HER-3, also known asErbB3) is a receptor protein tyrosine kinase and belongs to theepidermal growth factor receptor (EGFR) subfamily of receptor proteintyrosine kinases, which also includes HER-1 (also known as EGFR), HER-2,and HER-4 (Plowman et al., Proc. Natl. Acad. Sci. U.S.A. 87 (1990),4905-4909; Kraus et al., Proc. Natl. Acad. Sci. U.S.A. 86 (1989),9193-9197; and Kraus et al., Proc. Natl. Acad. Sci. U.S.A. 90 (1993),2900-2904). Like the prototypical epidermal growth factor receptor, thetransmembrane receptor HER-3 consists of an extracellular ligand-bindingdomain (ECD), a dimerization domain within the ECD, a transmembranedomain, an intracellular protein tyrosine kinase domain (TKD) and aC-terminal phosphorylation domain.

The ligand Heregulin (HRG) binds to the extracellular domain of HER-3and activates the receptor-mediated signaling pathway by promotingdimerization with other human epidermal growth factor receptor (HER)family members and transphosphorylation of its intracellular domain.Dimer formation between HER family members expands the signalingpotential of HER-3 and is a means not only for signal diversificationbut also signal amplification. For example the HER-2/HER-3 heterodimerinduces one of the most important mitogenic signals among HER familymembers.

HER-3 has been found to be overexpressed in several types of cancer suchas breast, gastrointestinal and pancreatic cancers. Interestingly acorrelation between the expression of HER-2/HER-3 and the progressionfrom a non-invasive to an invasive stage has been shown (Alimandi etal., Oncogene 10, 1813-1821; deFazio et al., Cancer 87, 487-498; Naiduet al., Br. J. Cancer 78, 1385-1390). Accordingly, agents that interferewith HER-3 mediated signaling are desirable. Murine or chimeric HER-3antibodies have been reported, such as in U.S. Pat. No. 5,968,511, U.S.Pat. No. 5,480,968 and WO03013602.

A humanized monoclonal antibody against HER-2, Herceptin®, has recentlybeen shown to interfere with HER-2 mediated signaling and istherapeutically effective in humans (Fendly et al., Hybridoma 6,359-370; Hudziak et al., Mol. Cell. Biol. 9, 1165-1172; Stebbing et al.,Cancer Treat Rev. 26, 287-290). Herceptin® has been shown to act throughtwo different mechanisms, i.e. the engagement of the effector cells ofthe immune system as well as a direct cytotoxic, apoptosis inducingeffect.

However, only patients with highly amplified HER-2 respond significantlyto Herceptin® therapy, thus limiting the number of patients suitable fortherapy. In addition the development of resistance to drugs or a changein the expression or epitope sequence of HER-2 on tumor cells may rendereven those approachable patients unreactive with the antibody andtherefore abrogating its therapeutic benefits. Therefore more drugs fortarget based therapies approaching further members of the HER family,such as HER-3, are needed.

BRIEF DESCRIPTION OF THE DRAWING FIGURES

FIG. 1 shows the extent of HER-3 expression in a panel of human cancercell lines and demonstrates that HER-3 is expressed in a variety ofhuman cancers.

FIG. 2 shows the results of the FACS analysis of HER-3 antibody bindingto either RatI cells stably expressing the different members of the HERfamily or only empty vector.

FIG. 3 shows antibody binding competition bins mapped to HER3 domains.

FIG. 4 show the results of the indirect FACS Scatchard antibody affinityanalysis performed with anti-HER-3 antibodies of the invention. Theanalysis indicates that the anti-HER-3 antibodies of the inventionpossess high affinities and strong binding constants for HER-3 expressedon the cell surface

FIG. 5 shows Anti-HER3 receptor antibody dependent HER3 receptorinternalization induced by anti-HER3 antibodies of the invention.

FIGS. 6 a-e show the results of a ligand competition assay performedwith anti-HER-3 antibodies of the invention. The results demonstratethat the antibodies of the invention specifically reduce binding of[¹²⁵I]-α-HRG/[¹²⁵I]-β-HRG to cells expressing endogenous HER-3.

FIG. 7 a shows the results of a HER-3 phosphotyrosine ELISA performedwith anti-HER-3 antibodies of the invention. Antibodies according to thepresent invention were able to inhibit β-HRG-mediated HER-3 activationas indicated by a decrease in receptor tyrosine phosphorylation.Furthermore FIG. 7 b shows representative results of this experimentwith titrated antibody.

FIG. 8 shows the result of a p42/p44 MAP-Kinase ELISA performed withanti-HER-3 antibodies of the invention. Antibodies according to thepresent invention were able to reduce β-HRG-mediated p42/p44 MAP-Kinaseactivation as indicated by increased MAP-Kinase phosphorylation.

FIG. 9 shows the result of a phospho-AKT ELISA performed with anti-HER-3antibodies of the invention. Antibodies according to the presentinvention were able to reduce β-HRG-mediated AKT activation as indicatedby AKT phosphorylation.

FIG. 10 shows the inhibition of MCF7 cell proliferation by humananti-HER-3 antibodies of the invention. Antibodies according to thepresent invention inhibit HRG-induced cell growth in human cancer cells.

FIG. 11 shows the transmigration of MCF7 cells inhibited by humananti-HER-3 antibodies of the invention.

FIGS. 12 a-i shows the inhibition of the anchorage independent cellgrowth by human HER-3 antibodies of the invention.

FIG. 13 shows the inhibition of xenograft growth of T47D human breastcancer cells by a human anti-HER-3 antibody of the invention.

FIG. 14 shows the reduction in xenograft growth of BxPC3 human pancreascancer cells in mice after administration of anti Her3 (U1-59 and U1-53)or anti EGFR (Erbitux) antibodies.

FIG. 15 shows the reduction of xenograft growth of BxPC3 human pancreascancer cells by a human anti-HER-3 antibody of the invention and incombination with anti EGFR (Erbitux) antibodies.

FIG. 16 demonstrates that antibodies of the invention delay xenograftgrowth of human melanoma (HT144) cell growth in nu/nu mice.

FIG. 17 shows the reduction of xenograft growth of HT-29 human coloncarcinoma cells by human HER-3 antibodies of the invention (U1-53, U1-59and U1-7).

FIG. 18 shows the reduction of xenograft growth of Calu-3 human lungcancer cells by human anti-HER-3 antibodies of the invention (U1-59,U1-53 and U1-7).

FIG. 19 shows the reduction of xenograft growth of BxPC-3 human pancreascancer cells by human anti-HER-3 antibodies of the invention (U1-7,U1-59 and U1-53).

FIG. 20 demonstrates that an antibody of the invention (U1-59) causessuppression of HER-3 in BxPC3 human pancreas cancer xenografts.

SUMMARY OF THE INVENTION

A first aspect of the present invention relates to an isolated bindingprotein that binds to HER-3.

In one embodiment of the present invention, an isolated binding proteinof the invention comprises a heavy chain amino acid sequence comprisingat least one of the CDR's selected from the group consisting of: (a)CDRH1's as shown in SEQ ID NOs:235-257, (b) CDRH2's as shown in SEQ IDNOs:258-282, and (c) CDRH3's as shown in SEQ ID NOs:283-317, and/or alight chain amino acid sequence comprising at least one of the CDR'sselected from the group consisting of: (d) CDRL1's as shown in SEQ IDNOs:318-342, (e) CDRL2's as shown in SEQ ID NOs:343-359, and (f) CDRL3'sas shown in SEQ ID NOs:360-388.

In another embodiment of the present invention, an isolated bindingprotein of the invention comprises a heavy chain amino acid sequenceselected from the group consisting of SEQ ID NOs: 2, 6, 10, 14, 18, 22,26, 30, 34, 36, 40, 42, 46, 50, 54, 60, 62, 66, 70, 74, 78, 80, 84, 88,92, 96, 100, 104, 108, 112, 116, 120, 122, 126, 130, 134, 138, 142, 146,150, 154, 158, 162, 166, 170, 174, 178, 182, 186, 190, 194, 198, 202,206, 210, 214, 218, 222, 226 and 230, and/or a light chain amino acidsequence selected from the group consisting of SEQ ID NOs: 4, 8, 12, 16,20, 24, 28, 32, 38, 44, 48, 52, 56, 58, 64, 68, 72, 76, 82, 86, 90, 94,98, 102, 106, 110, 114, 118, 124, 128, 132, 136, 140, 144, 148, 152,156, 160, 164, 168, 172, 176, 180, 184, 188, 192, 196, 200, 204, 208,212, 216, 220, 224, 228 and 232.

In yet another embodiment of the present invention, an isolated bindingprotein of the invention comprises a heavy chain amino acid sequence anda light chain amino acid sequence as shown in SEQ ID NOs: 2 and 4, 6 and8, 10 and 12, 14 and 16, 18 and 20, 22 and 24, 26 and 28, 30 and 32, 36and 38, 42 and 44, 46 and 48, 50 and 52, 54 and 56, 60 and 58, 62 and64, 66 and 68, 70 and 72, 74 and 76, 78 and 82, 80 and 82, 84 and 86, 88and 90, 92 and 94, 96 and 98, 100 and 102, 104 and 106, 108 and 110, 112and 114, 116 and 118, 122 and 124, 126 and 128, 130 and 132, 134 and136, 138 and 140, 142 and 144, 146 and 148, 150 and 152, 154 and 156,158 and 160, 162 and 164, 166 and 168, 170 and 172, 174 and 176, 178 and180, 182 and 184, 186 and 188, 190 and 192, 194 and 196, 198 and 200,202 and 204, 206 and 208, 210 and 212, 214 and 216, 218 and 220, 222 and224, 226 and 228, 230 and 232, or a heavy chain amino acid sequence asshown in SEQ ID NOs: 34, 40, 60, 62 or 120, or a light chain amino acidsequence as shown in SEQ ID NOs: 58 or 64.

According to the present invention, an isolated binding protein that iscapable of binding to HER-3 interacts with at least one epitope in theextracellular part of HER-3 (SEQ ID NO:390). The epitopes are preferablylocated in domain L1 (aa) 19-184 of SEQ ID NO:390) which is the aminoterminal domain, in domain S1 (aa) 185-327 of SEQ ID NO:390) and S2 (aa)500-632 of SEQ ID NO:390) which are the two Cysteine-rich domains, or indomain L2 (aa 328-499 of SEQ ID NO:390) which is flanked by the twoCysteine-rich domains. The epitopes may also be located in combinationsof domains such as but not limited to an epitope comprised by parts ofL1 and S1. Further preferred is an isolated binding protein that bindsto a three-dimensional structure formed by amino acid residues 1-160,161-358, 359-575, 1-358 and/or 359-604 of mature HER-3 (SEQ ID NO:390),particularly of mature human HER-3.

Preferably, an isolated binding protein of the invention is a scaffoldprotein having an antibody like binding activity or an antibody, e.g. ananti-HER-3 antibody. In particular, the anti-HER-3 antibody is selectedfrom the group consisting of U1-1 antibody, U1-2-antibody, U1-3antibody, U1-4 antibody, U1-5 antibody, U1-6 antibody, U1-7 antibody,U1-8 antibody, U1-9 antibody, U1-10 antibody, U1-11 antibody, U1-12antibody, U1-13 antibody, U1-14 antibody, U1-15 antibody, U1-16antibody, U1-17 antibody, U1-18 antibody, U1-19 antibody, U1-20antibody, U1-21 antibody, U1-22 antibody, U1-23 antibody, U1-24antibody, U1-25 antibody, U1-26 antibody, U1-27 antibody, U1-28antibody, U1-29 antibody, U1-30 antibody, U1-31 antibody, U1-32antibody, U1-33 antibody, U1-34 antibody, U1-35 antibody, U1-36antibody, U1-37 antibody, U1-38 antibody, U1-39 antibody, U1-40antibody, U1-41 antibody, U1-42 antibody, U1-43 antibody, U1-44antibody, U1-45 antibody, U1-46 antibody, U1-47 antibody, U148 antibody,U1-49 antibody, U1-50 antibody, U1-51 antibody, U1-52 antibody, U1-53antibody, U1-55.1 antibody, U1-55 antibody, U1-57.1 antibody, U1-57antibody, U1-58 antibody, U1-59 antibody, U161.1 antibody, U1-61antibody, U1-62 antibody or an antibody having at least one heavy orlight chain of one of said antibodies. Especially preferred are theantibodies U1-49 (SEQ ID NO: 42/44), U1-53 (SEQ ID NO: 54/56) and U1-59(SEQ ID NO: 70/72) or an antibody having at least one heavy or lightchain of one of said antibodies.

In addition, further embodiments of the present invention provide anisolated binding protein coupled to a labelling group or effector group.Preferably, such an binding protein is useful for the treatment ofhyperproliferative diseases, particularly oncological diseases such asbreast cancer, gastrointestinal cancer, pancreatic cancer, prostatecancer, ovarian cancer, stomach cancer, endometrial cancer, salivarygland cancer, lung cancer, kidney cancer, colon cancer, colorectalcancer, thyroid cancer, bladder cancer, glioma, melanoma, testis cancer,soft tissue sarcoma, head and neck cancer, or other HER-3 expressing oroverexpressing cancers, and the formation of tumor metastases.

Other aspects of the present invention relate to an isolated nucleicacid molecule encoding a binding protein of the invention, a vectorhaving a nucleic acid molecule encoding the binding protein of theinvention, and a host cell, e.g. a CHO cell, an NS/0 myeloma cell,transformed with such nucleic acid molecule or vector.

A further aspect of the present invention relates to a method forproducing a binding protein of the invention by preparing said bindingprotein from a host cell that secretes the binding protein. Preferably,the binding protein of the invention is prepared from a hybridoma cellline that secretes a binding protein or a CHO or other cell typetransformed with a nucleic acid molecule encoding a binding protein ofthe invention.

Another aspect of the present invention relates to a method forproducing a binding protein of the invention by preparing said bindingprotein from a tissue, product or secretion of an animal, plant orfungus transgenic for a nucleic acid molecule or nucleic acid moleculesencoding the binding protein of the invention. Preferably, a bindingprotein of the invention is prepared from the tissue, product orsecretion of a transgenic animal such as cow, sheep, rabbit, chicken orother mammalian or avian species, a transgenic plant such as corn,tobacco or other plant, or a transgenic fungus such as Aspergillus,Pichia or other fungal species.

Another aspect of the present invention pertains to a pharmaceuticalcomposition comprising as an active agent at least one binding proteinof the invention in admixture with pharmaceutically acceptable carriers,diluents and/or adjuvants. In another preferred embodiment of thepresent invention, the pharmaceutical composition of the inventionadditionally contains at least one other active agent, e.g. at least oneantineoplastic agent. Yet another aspect of the present inventionpertains to the use of at least one binding protein of the invention,and optionally at least one other active agent, e.g. at least oneantineoplastic agent, in admixture with pharmaceutically acceptablecarriers, diluents and/or adjuvants for the preparation of apharmaceutical composition. The pharmaceutical composition is suitablefor diagnosing, preventing or treating a hyperproliferative disease,particularly an oncological disease such as breast cancer,gastrointestinal cancer, pancreas cancer, prostate cancer, ovariancancer, stomach cancer, endometrial cancer, salivary gland cancer, lungcancer, kidney cancer, colon cancer, colorectal cancer, thyroid cancer,bladder cancer, glioma, melanoma or other HER-3 expressing oroverexpressing cancers, and the formation of tumor metastases.

Moreover, the present invention relates in a further aspect to a methodfor diagnosing diseases or conditions associated with the expression ofHER-3, comprising contacting a sample with at least one binding proteinof the invention, and detecting the presence of HER-3. Preferreddiseases or conditions include the hyperproliferative diseases mentionedabove.

Still another aspect of the present invention is a method for preventingor treating diseases or conditions associated with the expression ofHER-3 in a patient in need thereof, comprising administering to thepatient an effective amount of at least one binding protein of theinvention and optionally at least one other active agent, e.g. at leastone neoplastic agent. Preferably, the patient is a mammalian patient,more preferably a human patient. Preferred diseases or conditionsassociated with the expression of HER-3 are the hyperproliferativediseases mentioned above.

A further aspect of the present invention relates to a kit for thediagnosis, prevention or treatment diseases or conditions associatedwith the expression of HER-3, comprising at least one binding protein,and/or nucleic acid molecule and/or vector of the invention. Optionally,the kit of the invention can further comprise at least one other activeagent, e.g. at least one anti neoplastic agent. Preferably, the diseasesor conditions associated with the expression of HER-3 are thehyperproliferative diseases mentioned above.

DETAILED DESCRIPTION

A first aspect of the present invention relates to an isolated bindingprotein that binds to HER-3.

In one embodiment of the present invention, the isolated binding proteinof the invention comprises a heavy chain amino acid sequence comprisingat least one of the CDR's selected from the group consisting of: (a)CDRH1's as shown in SEQ ID NOs:235-257, (b) CDRH2's as shown in SEQ IDNOs:258-282, and (c) CDRH3's as shown in SEQ ID NOs:282-317, and/or alight chain amino acid sequence comprising at least one of the CDR'sselected from the group consisting of: (d) CDRL1's as shown in SEQ IDNOs:318-342, (e) CDRL2's as shown in SEQ ID NOs:343-359, and (f) CDRL3'sas shown in SEQ ID NOs:360-388.

In another embodiment of the present invention, the isolated bindingprotein of the invention comprises a heavy chain amino acid sequenceselected from the group consisting of SEQ ID NOs: 2, 6, 10, 14, 18, 22,26, 30, 34, 36, 40, 42, 46, 50, 54, 60, 62, 66, 70, 74, 78, 80, 84, 88,92, 96, 100, 104, 108, 112, 116, 120, 122, 126, 130, 134, 138, 142, 146,150, 154, 158, 162, 166, 170, 174, 178, 182, 186, 190, 194, 198, 202,206, 210, 214, 218, 222, 226 and 230, and/or a light chain amino acidsequence selected from the group consisting of SEQ ID NOs: 4, 8, 12, 16,20, 24, 28, 32, 38, 44, 48, 52, 56, 58, 64, 68, 72, 76, 82, 86, 90, 94,98, 102, 106, 110, 114, 118, 124, 128, 132, 136, 140, 144, 148, 152,156, 160, 164, 168, 172, 176, 180, 184, 188, 192, 196, 200, 204, 208,212, 216, 220, 224, 228 and 232.

In yet another embodiment of the present invention, the isolated bindingprotein of the invention comprises a heavy chain amino acid sequence anda light chain amino acid sequence as shown in SEQ ID NOs: 2 and 4, 6 and8, 10 and 12, 14 and 16, 18 and 20, 22 and 24, 26 and 28, 30 and 32, 36and 38, 42 and 44, 46 and 48, 50 and 52, 54 and 56, 60 and 58, 62 and64, 66 and 68, 70 and 72, 74 and 76, 78 and 82, 80 and 82, 84 and 86, 88and 90, 92 and 94, 96 and 98, 100 and 102, 104 and 106, 108 and 110, 112and 114, 116 and 118, 122 and 124, 126 and 128, 130 and 132, 134 and136, 138 and 140, 142 and 144, 146 and 148, 150 and 152, 154 and 156,158 and 160, 162 and 164, 166 and 168, 170 and 172, 174 and 176, 178 and180, 182 and 184, 186 and 188, 190 and 192, 194 and 196, 198 and 200,202 and 204, 206 and 208, 210 and 212, 214 and 216, 218 and 220, 222 and224, 226 and 228, 230 and 232, or a heavy chain amino acid sequence asshown in SEQ ID NOs: 34, 40, 60, 62 or 120, or a light chain amino acidsequence as shown in SEQ ID NOs: 58 or 64.

In accordance with the present invention, it is to be understood, thatthe amino acid sequence of the binding protein of the invention is notlimited to the twenty conventional amino acids (See Immunology—ASynthesis (2^(nd) Edition, E. S. Golub and D. R. Gren, Eds., SinauerAssociates, Sunderland, Mass. (1991)), which is incorporated herein byreference). For example, the amino acids may include stereoisomers (e.g.D-amino acids) of the twenty conventional amino acids, unnatural aminoacids such as α-,α-disubstituted amino acids, N-alkyl amino acids,lactic acid, and other unconventional amino acids. Examples ofunconventional amino acids, which may also be suitable components forthe binding protein of the invention, include: 4-hydroxyproline,γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine,O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine,5-hydroxylysine, σ-N-methylarginine, and other similar amino acids andimino acids, e.g. 4-hydroxyproline.

Furthermore, in accordance with the present invention, minor variationsin the amino acid sequences shown in SEQ ID NOs:2, 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50,52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86,88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116,118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144,146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172,174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200,202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228,230 and 232 are contemplated as being encompassed by the presentinvention, providing that the variations in the amino acid sequencemaintain at least 75%, more preferably at least 80%, 90%, 95%, and mostpreferably 99% of the sequences shown in SEQ ID NOs:2, 4, 6, 8, 10, 12,14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48,50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84,86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116,118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144,146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172,174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200,202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228,230 and 232. The variations may occur within the framework regions (i.e.outside the CDRs), within the CDRs, or within the framework regions andthe CDRs. Preferred variations in the amino acid sequences shown in SEQID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70,72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104,106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132,134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160,162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188,190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216,218, 220, 222, 224, 226, 228, 230 and 232, i.e. deletions, insertionsand/or replacements of at least one amino acid, occur near boundaries offunctional domains. Structural and functional domains can be identifiedby comparison of the nucleotide and/or amino acid sequence data topublic or proprietary sequence databases. Computerized comparisonmethods can be used to identify sequence motifs or predicted proteinconformation domains that occur in other binding proteins of knownstructure and/or function. Methods to identify protein sequences thatfold into a known three-dimensional structure are known. See e.g. Bowieet al., Science 253, 164 (1991); Proteins, Structures and MolecularPrinciples (Creighton, Ed., W. H. Freeman and Company, New York (1984));Introduction to Protein Structure (C. Branden and J. Tooze, eds.,Garland Publishing, New York, N.Y. (1991)); and Thornton et al., Nature354, 105 (1991), which are all incorporated herein by reference. Thus,those of skill in the art can recognize sequence motifs and structuralconformations that may be used to define structural and functionaldomains in accordance with the invention.

Especially preferred variations in the amino acid sequences shown in SEQID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70,72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104,106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132,134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160,162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188,190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216,218, 220, 222, 224, 226, 228, 230 and 232, are those that lead to areduced susceptibility to proteolysis or oxidation, alter glycosylationpatterns or alter binding affinities or confer or modify otherphysicochemical or functional properties of the binding protein. Inparticular, conservative amino acid replacements are contemplated.Conservative replacements are those that take place within a family ofamino acids that are related in their side chains. Preferred amino acidfamilies are the following: acidic family=aspartate, glutamate; basicfamily=lysine, arginine, histidine; non-polar family=alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; anduncharged polar family=glycine, asparagine, glutamine, cysteine, serine,threonine, tyrosine. More preferred families are: aliphatic-hydroxyfamily=serine and threonine; amide-containing family=asparagine andglutamine; aliphatic family=alanine, valine, leucine and isoleucine; andaromatic family=phenylalanine, tryptophan, and tyrosine. For example, itis reasonable to expect that an isolated replacement of a leucine withan isoleucine or valine, an aspartate with a glutamate, a threonine witha serine, or a similar replacement of an amino acid with a structurallyrelated amino acid will not have a major effect on the binding orproperties of the resulting binding protein, especially if thereplacement does not involve an amino acid within a framework site.However, all other possible amino acid replacements are also encompassedby the present invention. Whether an amino acid change results in afunctional binding protein, i.e. in a binding protein that binds toHER-3 and reduces signal transduction of HER family members, can readilybe determined by assaying the specific activity of the resulting bindingprotein in ELISA or FACS for binding to HER-3 or in vitro or in vivofunctional assay.

According to the present invention, the binding protein of the inventioninteracts with at least one epitope in the extracellular part of HER-3(SEQ ID NO:390). The epitopes are preferably located in domain L1 (aa )19-184 of SEQ ID NO:390) which is the amino terminal domain, in domainS1 (aa) 185-327 of SEQ ID NO:390) and S2 (aa) 500-632 of SEQ ID NO:390)which are the two Cysteine-rich domains, in domain L2 (aa 328-499 of SEQID NO:390) which is flanked by the two Cysteine-rich domains, or in acombination of HER-3 domains. The epitopes may also be located incombinations of domains such as but not limited to an epitope comprisedby parts of L1 and S1. Moreover, the binding protein of the invention isfurther characterized in that its binding to HER-3 reducesHER-3-mediated signal transduction. In accordance with the presentinvention, a reduction of HER-3-mediated signal transduction may, e.g.be caused by a downregulation of HER-3 resulting in an at least partialdisappearance of HER-3 molecules from the cell surface or by astabilization of HER-3 on the cell surface in a substantially inactiveform, i.e. a form which exhibits a lower signal transduction compared tothe non-stabilized form. Alternatively, a reduction of HER-3-mediatedsignal transduction may also be caused by influencing, e.g. decreasingor inhibiting, the binding of a ligand or another member of the HERfamily to HER-3, of GRB2 to HER-2 or of GRB2 to SHC, by inhibitingreceptor tyrosine phosphorylation, AKT phosphorylation, PYK2 tyrosinephosphorylation or ERK2 phosphorylation, or by decreasing tumorinvasiveness. Alternatively, a reduction of HER-3 mediated signaltransduction may also be caused by influencing, e.g., decreasing orinhibiting, the formation of HER-3 containing dimers with other HERfamily members. One example among others may be the decreasing orinhibiting of the HER3-EGFR protein complex formation.

Preferably, the binding protein of the invention is a scaffold proteinhaving an antibody like binding activity or an antibody, i.e. ananti-HER-3 antibody.

Within the context of the present invention, the term “scaffoldprotein”, as used herein, means a polypeptide or protein with exposedsurface areas in which amino acid insertions, substitutions or deletionsare highly tolerable. Examples of scaffold proteins that can be used inaccordance with the present invention are protein A from Staphylococcusaureus, the bilin binding protein from Pieris brassicae or otherlipocalins, ankyrin repeat proteins, and human fibronectin (reviewed inBinz and Plückthun, Curr Opin Biotechnol, 16, 459-69). Engineering of ascaffold protein can be regarded as grafting or integrating an affinityfunction onto or into the structural framework of a stably foldedprotein. Affinity function means a protein binding affinity according tothe present invention. A scaffold can be structurally separable from theamino acid sequences conferring binding specificity. In general,proteins appearing suitable for the development of such artificialaffinity reagents may be obtained by rational, or most commonly,combinatorial protein engineering techniques such as panning againstHER-3, either purified protein or protein displayed on the cell surface,for binding agents in an artificial scaffold library displayed in vitro,skills which are known in the art (Skerra, J. Mol. Recog., 2000; Binzand Plückthun, 2005). In addition, a scaffold protein having an antibodylike binding activity can be derived from an acceptor polypeptidecontaining the scaffold domain, which can be grafted with bindingdomains of a donor polypeptide to confer the binding specificity of thedonor polypeptide onto the scaffold domain containing the acceptorpolypeptide. Said inserted binding domains may be, for example, thecomplementarity determining region (CDR) of an antibody, in particularan anti-HER-3 antibody. Insertion can be accomplished by various methodsknown to those skilled in the art including, for example, polypeptidesynthesis, nucleic acid synthesis of an encoding amino acid as well byvarious forms of recombinant methods well known to those skilled in theart.

Moreover, the term “antibody” or “anti-HER-3 antibody”, as used herein,means a monoclonal antibody, a polyclonal antibody, a recombinantantibody, a humanized antibody (Jones et al., Nature 321 (1986),522-525; Riechmann et al., Nature 332 (1988), 323-329; and Presta, Curr.Op. Struct. Biol. 2 (1992), 593-596), a chimeric antibody (Morrison etal., Proc. Natl. Acad. Sci. U.S.A. 81 (1984), 6851-6855), amultispecific antibody (e.g. a bispecific antibody) formed from at leasttwo antibodies, or an antibody fragment thereof. The term “antibodyfragment” comprises any portion of the afore-mentioned antibodies,preferably their antigen binding or variable regions. Examples ofantibody fragments include Fab fragments, Fab′ fragments, F(ab′)₂fragments, Fv fragments, diabodies (Hollinger et al., Proc. Natl. Aced.Sci. U.S.A. 90 (1993), 6444-6448), single chain antibody molecules(Pluckthun in: The Pharmacology of Monoclonal Antibodies 113, Rosenburgand Moore, EDS, Springer Verlag, N.Y. (1994), 269-315) and otherfragments as long as they exhibit the desired capability of binding toHER-3.

In addition, the term “antibody” or “anti-HER-3 antibody”, as usedherein, may include antibody-like molecules that contain engineeredsub-domains of antibodies or naturally occurring antibody variants.These antibody-like molecules may be single-domain antibodies such asVH-only or VL-only domains derived either from natural sources such ascamelids (Muyldermans et al., Reviews in Molecular Biotechnology 74,277-302) or through in vitro display of libraries from humans, camelidsor other species (Holt et al., Trends Biotechnol., 21, 484-90).

In accordance with the present invention, the “Fv fragment” is theminimum antibody fragment that contains a complete antigen-recognitionand -binding site. This region consists of a dimer of one heavy- and onelight-chain variable domain in tight, non-covalent association. It is inthis configuration that the three CDR's of each variable domain interactto define an antigen-binding site on the surface of the V_(H)-V_(L)dimer. Collectively, the six CDR's confer antigen-binding specificity tothe antibody. However, even a single variable domain (or half of an Fvcomprising only three CDR's specific for an antigen) has the ability torecognize and bind the antigen, although usually at a lower affinitythan the entire binding site. The “Fab fragment” also contains theconstant domain of the light chain and the first constant domain (CH1)of the heavy chain. The “Fab fragment” differs from the “Fab′ fragment”by the addition of a few residues at the carboxy terminus of the heavychain CH1 domain including one or more cysteines from the antibody hingeregion. The “F(ab′)₂ fragment” originally is produced as a pair of “Fab′fragments” which have hinge cysteines between them. Methods of preparingsuch antibody fragments, such as papain or pepsin digestion, are knownto those skilled in the art.

In a preferred embodiment of the present invention, the anti-HER-3antibody of the invention is of the IgA-, IgD-, IgE, IgG- or IgM-type,preferably of the IgG- or IgM-type including, but not limited to, theIgG1-, IgG2-, IgG3-, IgG4-, IgM1- and IgM2-type. In most preferredembodiments, the antibody is of the IgG1-, IgG2- or IgG4-type.

In another preferred embodiment of the present invention, the anti-HER-3antibody of the invention is an anti-HER-3 antibody directed against theextracellular domain (ECD) of HER-3.

In certain respects, e.g. in connection with the generation ofantibodies as therapeutic candidates against HER-3, it may be desirablethat the anti-HER-3 antibody of the invention is capable of fixingcomplement and participating in complement-dependent cytotoxicity (CDC).There are a number of isotypes of antibodies that are capable of thesame including without limitations the following: murine IgM, murineIgG2a, murine IgG2b, murine IgG3, human IgM, human IgG1, human IgG3, andhuman IgA. It will be appreciated that antibodies that are generatedneed not initially possess such an isotype but, rather the antibody asgenerated can possess any isotype and the antibody can be isotypeswitched by appending the molecularly cloned V region genes or cDNA tomolecularly cloned constant region genes or cDNAs in appropriateexpression vectors using conventional molecular biological techniquesthat are well known in the art and then expressing the antibodies inhost cells using techniques known in the art. The isotype-switchedantibody may also possess an Fc region that has been molecularlyengineered to possess superior CDC over naturally occurring variants(Idusogie et al., J Immunol., 166, 2571-2575) and expressedrecombinantly in host cells using techniques known in the art. Suchtechniques include the use of direct recombinant techniques (see e.g.U.S. Pat. No. 4,816,397), cell-cell fusion techniques (see e.g. U.S.Pat. Nos. 5,916,771 and 6,207,418), among others. In the cell-cellfusion technique, a myeloma or other cell line such as CHO is preparedthat possesses a heavy chain with any desired isotype and anothermyeloma or other cell line such as CHO is prepared that possesses thelight chain. Such cells can, thereafter, be fused and a cell lineexpressing an intact antibody can be isolated. By way of example, ahuman anti-HER-3 IgG4 antibody, that possesses the desired binding tothe HER-3 antigen, could be readily isotype switched to generate a humanIgM, human IgG1 or human IgG3 isotype, while still possessing the samevariable region (which defines the antibody's specificity and some ofits affinity). Such molecule might then be capable of fixing complementand participating in CDC.

Moreover, it may also be desirable for the anti-HER-3 antibody of theinvention to be capable of binding to Fc receptors on effector cells,such as monocytes and natural killer (NK) cells, and participate inantibody-dependent cellular cytotoxicity (ADCC). There are a number ofisotypes of antibodies that are capable of the same, including withoutlimitations the following: murine IgG2a, murine IgG2b, murine IgG3,human IgG1 and human IgG3. It will be appreciated that antibodies thatare generated need not initially possess such an isotype but, rather theantibody as generated can possess any isotype and the antibody can beisotype switched by appending the molecularly cloned V region genes orcDNA to molecularly cloned constant region genes or cDNAs in appropriateexpression vectors using conventional molecular biological techniquesthat are well known in the art and then expressing the antibodies inhost cells using techniques known in the art. The isotype-switchedantibody may also possess an Fc region that has been molecularlyengineered to possess superior ADCC over naturally occurring variants(Shields et al. J Biol Chem., 276, 6591-6604) and expressedrecombinantly in host cells using techniques known in the art. Suchtechniques include the use of direct recombinant techniques (see e.g.U.S. Pat. No. 4,816,397), cell-cell fusion techniques (see e.g. U.S.Pat. Nos. 5,916,771 and 6,207,418), among others. In the cell-cellfusion technique, a myeloma or other cell line such as CHO is preparedthat possesses a heavy chain with any desired isotype and anothermyeloma or other cell line such as CHO is prepared that possesses thelight chain. Such cells can, thereafter, be fused and a cell lineexpressing an intact antibody can be isolated. By way of example, ahuman anti-HER-3 IgG4 antibody, that possesses the desired binding tothe HER-3 antigen, could be readily isotype switched to generate a humanIgG1 or human IgG3 isotype, while still possessing the same variableregion (which defines the antibody's specificity and some of itsaffinity). Such molecule might then be capable of binding to FcγR oneffectors cells and participating in ADCC.

Furthermore, according to the present invention, it is appreciated thatthe anti-HER-3 antibody of the invention is a fully human or humanizedantibody. Human antibodies avoid certain of the problems associated withxenogeneic antibodies, for example antibodies that possess murine or ratvariable and/or constant regions. The presence of xenogeneic-derivedproteins such murine or rat derived proteins can lead to the generationof an immune response against the antibody by a patient, subsequentlyleading to the rapid clearance of the antibodies, loss of therapeuticutility through neutralization of the antibody and/or severe, evenlife-threatening, allergic reactions.

Preferably, the anti-HER-3 antibody of the invention is selected fromthe group consisting of U1-1 antibody, U1-2 antibody, U1-3 antibody,U1-4 antibody, U1-5 antibody, U1-6 antibody, U1-7 antibody, U1-8antibody, U1-9 antibody, U1-10 antibody, U1-11 antibody, U1-12 antibody,U1-13 antibody, U1-14 antibody, U1-15 antibody, U1-16 antibody, U1-17antibody, U1-18 antibody, U1-19 antibody, U1-20 antibody, U1-21antibody, U1-22 antibody, U1-23 antibody, U1-24 antibody, U1-25antibody, U1-26 antibody, U1-27 antibody, U1-28 antibody, U1-29antibody, U1-30 antibody, U1-31 antibody, U1-32 antibody, U1-33antibody, U1-34 antibody, U1-35 antibody, U1-36 antibody, U1-37antibody, U1-38 antibody, U1-39 antibody, U1-40 antibody, U1-41antibody, U1-42 antibody, U1-43 antibody, U1-44 antibody, U1-45antibody, U1-46 antibody, U1-47 antibody, U1-48 antibody, U1-49antibody, U1-50 antibody, U1-51 antibody, U1-52 antibody, U1-53antibody, U1-55.1 antibody, U1-55 antibody, U1-57.1 antibody, U1-57antibody, U1-58 antibody, U1-59 antibody, U1-61.1 antibody, U1-61antibody, U1-62 antibody.

In a preferred embodiment of the present invention, a binding protein ofthe invention is coupled to a labelling group. Such a binding protein isparticularly suitable for diagnostic applications. As used herein, theterm “labelling group” refers to a detectable marker, e.g. aradiolabelled amino acid or biotinyl moiety that can be detected bymarked avidin (e.g. streptavidin bound to a fluorescent marker orenzymatic activity that can be detected by optical or colorimetricmethods). Various methods for labelling polypeptides and glycoproteins,such as antibodies, are known in the art and may be used in performingthe present invention. Examples of suitable labelling groups include,but are not limited to, the following: radioisotopes or radionuclides(e.g. ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I), fluorescentgroups (e.g. FITC, rhodamine, lanthanide phosphors), enzymatic groups(e.g. horseradish peroxidase, β-galactosidase, luciferase, alkalinephosphatase), chemiluminescent groups, biotinyl groups, or predeterminedpolypeptide epitopes recognized by a secondary reporter (e.g. leucinezipper pair sequences, binding sites for secondary antibodies, metalbinding domains, epitope tags). In certain respects, it may be desirablethat the labelling groups are attached by spacer arms of various lengthsto reduce potential steric hindrance.

Alternatively, a binding protein of the invention may be coupled to aneffector group in another preferred embodiment of the invention. Such abinding protein is especially suitable for therapeutic applications. Asused herein, the term “effector group” refers to a cytotoxic group suchas a radioisotope or radionuclide, a toxin, a therapeutic group or othereffector group known in the art. Examples for suitable effector groupsare radioisotopes or radionuclides (e.g. ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc,¹¹¹In, ¹²⁵I, ¹³¹I), calicheamicin, dolastatin analogs such asauristatins, and chemotherapeutic agents such as geldanamycin andmaytansine derivates, including DM1. In certain respects, it may bedesirable that the effector groups are attached by spacer arms ofvarious lengths to reduce potential steric hindrance.

A second aspect of the present invention relates to a process forpreparing an isolated binding protein of the invention, comprising thestep of preparing the binding protein from a host cell that secretes thebinding protein. Host cells, that may be used according to the presentinvention, are hybridomas; eukaryotic cells such as mammalian cells,e.g. hamster, rabbit, rat, pig, mouse or other animal cells, plantcells, fungal cells, e.g. Saccharomyces cerevisiae, Pichia pastoris;prokaryotic cells such as E. coli; and other cells used in the art forthe production of binding proteins. Various methods for preparing andisolating binding proteins, such as scaffold proteins or antibodies,from host cells are known in the art and may be used in performing thepresent invention. Moreover, methods for preparing binding proteinfragments, e.g. scaffold protein fragments or antibody fragments, suchas papain or pepsin digestion, modern cloning techniques, techniques forpreparing single chain antibody molecules (Plückthun in: ThePharmacology of Monoclonal Antibodies 113, Rosenburg and Moore, EDS,Springer Verlag, N.Y. (1994), 269-315) and diabodies (Hollinger et al.,Proc. Natl. Acad. Sci. U.S.A. 90 (1993), 6444-6448), are also known tothose skilled in the art and may be used in performing the presentinvention.

In a preferred embodiment of the present invention, a binding protein ofthe invention is prepared from a hybridoma that secretes the bindingprotein. See e.g. Köhler et al., Nature 256 (1975), 495.

In a further preferred embodiment of the present invention, a bindingprotein of the invention is prepared recombinantly optimizing and/oramplifying expression of the binding protein in a host cell andisolating the binding protein from said host cell. To this end, the hostcells are transformed or transfected with DNA encoding a binding proteinor a vector containing DNA encoding the binding protein and culturedunder appropriate conditions to produce the binding protein of theinvention. See e.g. U.S. Pat. No. 4,816,567. Preferred host cells may beCHO cells, NS/0 myeloma cells, human embryonic kidney 293 cells, E. coliand Saccharomyces cerevisiae.

With regard to binding proteins that are antibodies, these antibodiesmay be prepared from animals genetically engineered to make fully humanantibodies or from an antibody display library made in bacteriophage,yeast, ribosome or E. coli. See e.g. Clackson et al., Nature 352 (1991),624-628, Marks et al., J. Mol. Biol. 222 (1991), 581-597, Feldhaus andSiegel J Immunol Methods. 290, 69-80, Groves and Osbourn, Expert OpinBiol Ther., 5, 125-135 and Jostock and Dubel, Comb Chem High ThroughputScreen. 8, 127-133.

Human antibodies avoid some of the problems associated with antibodiesthat possess murine or rat variable and/or constant regions. Thepresence of such murine or rat derived proteins can lead to the rapidclearance of the antibodies or can lead to the generation of an immuneresponse against the antibody by a patient. In order to avoid theutilization of murine or rat derived antibodies, fully human antibodiescan be generated through the introduction of functional human antibodyloci into a rodent, other mammal or animal so that the rodent, othermammal or animal produces fully human antibodies.

One method for generating fully human antibodies is through the use ofXENOMOUSE® strains of mice that have been engineered to contain 245 kband 190 kb-sized germline configuration fragments of the human heavychain locus and kappa light chain locus. Other XenoMouse strains of micecontain 980 kb and 800 kb-sized germline configuration fragments of thehuman heavy chain locus and kappa light chain locus. Still otherXenoMouse strains of mice contain 980 kb and 800 kb-sized germlineconfiguration fragments of the human heavy chain locus and kappa lightchain locus plus a 740 kb-sized germline configured complete humanlambda light chain locus. See Mendez et al. Nature Genetics 15:146-156(1997) and Green and Jakobovits J. Exp. Med. 188:483-495 (1998). TheXENOMOUSE® strains are available from Abgenix, Inc. (Fremont, Calif.).

The production of the XENOMOUSE® mice is further discussed anddelineated in U.S. patent application Ser. No. 07/466,008, filed Jan.12, 1990, Ser. No. 07/610,515, filed Nov. 8, 1990, Ser. No. 07/919,297,filed Jul. 24, 1992, Ser. No. 07/922,649, filed Jul. 30, 1992, filedSer. No. 08/031,801, filed Mar. 15, 1993, Ser. No. 08/112,848, filedAug. 27, 1993, Ser. No. 08/234,145, filed Apr. 28, 1994, Ser. No.08/376,279, filed Jan. 20, 1995, Ser. No. 08/430,938, Apr. 27, 1995,Ser. No. 08/464,584, filed Jun. 5, 1995, Ser. No. 08/464,582, filed Jun.5, 1995, Ser. No. 08/463,191, filed Jun. 5, 1995, Ser. No. 08/462,837,filed Jun. 5, 1995, Ser. No. 08/486,853, filed Jun. 5, 1995, Ser. No.08/486,857, filed Jun. 5, 1995, Ser. No. 08/486,859, filed Jun. 5, 1995,Ser. No. 08/462,513, filed Jun. 5, 1995, Ser. No. 08/724,752, filed Oct.2, 1996, and Ser. No. 08/759,620, filed Dec. 3, 1996, U.S. PatentPublication 2003/0217373, filed Nov. 20, 2002, and U.S. Pat. Nos.6,833,268, 6,162,963, 6,150,584, 6,114,598, 6,075,181, and 5,939,598 andJapanese Patent Nos. 3 068 180 B2, 3 068 506 B2, and 3 068 507 B2. Seealso European Patent No. EP 0 463 151 B1, grant published Jun. 12, 1996,International Patent Application No. WO 94/02602, published Feb. 3,1994, International Patent Application No. WO 96/34096, published Oct.31, 1996, WO 98/24893, published Jun. 11, 1998, WO 00/76310, publishedDec. 21, 2000. The disclosures of each of the above-cited patents,applications, and references are hereby incorporated by reference intheir entirety.

In an alternative approach, others, including GenPharm International,Inc., have utilized a “minilocus” approach. In the minilocus approach,an exogenous Ig locus is mimicked through the inclusion of pieces(individual genes) from the Ig locus. Thus, one or more V_(H) genes, oneor more DH genes, one or more J_(H) genes, a mu constant region, and asecond constant region (preferably a gamma constant region) are formedinto a construct for insertion into an animal. This approach isdescribed in U.S. Pat. No. 5,545,807 to Surani et al. and U.S. Pat. Nos.5,545,806, 5,625,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429,5,789,650, 5,814,318, 5,877,397, 5,874,299, and 6,255,458 each toLonberg and Kay, U.S. Pat. Nos. 5,591,669 and 6,023.010 to Krimpenfortand Berns, U.S. Pat. Nos. 5,612,205, 5,721,367, and 5,789,215 to Bernset al., and U.S. Pat. No. 5,643,763 to Choi and Dunn, and GenPharmInternational U.S. patent application Ser. No. 07/574,748, filed Aug.29, 1990, Ser. No. 07/575,962, filed Aug. 31, 1990, Ser. No. 07/810,279,filed Dec. 17, 1991, Ser. No. 07/853,408, filed Mar. 18, 1992, Ser. No.07/904,068, filed Jun. 23, 1992, Ser. No. 07/990,860, filed Dec. 16,1992, Ser. No. 08/053,131, filed Apr. 26, 1993, Ser. No. 08/096,762,filed Jul. 22, 1993, Ser. No. 08/155,301, filed Nov. 18, 1993, Ser. No.08/161,739, filed Dec. 3, 1993, Ser. No. 08/165,699, filed Dec. 10,1993, Ser. No. 08/209,741, filed Mar. 9, 1994, the disclosures of whichare hereby incorporated by reference. See also European Patent No. 0 546073 B1, International Patent Application Nos. WO 92/03918, WO 92/22645,WO 92/22647, WO 92/22670, WO 93/12227, WO 94/00569, WO 94/25585, WO96/14436, WO 97/13852, and WO 98/24884 and U.S. Pat. No. 5,981,175, thedisclosures of which are hereby incorporated by reference in theirentirety. See further Taylor et al., 1992, Chen et al., 1993, Tuaillonet al., 1993, Choi et al, 1993, Lonberg et al., (1994), Taylor et al.,(1994), and Tuaillon et al., (1995), Fishwild et al., (1996), thedisclosures of which are hereby incorporated by reference in theirentirety.

Kirin has also demonstrated the generation of human antibodies from micein which, through microcell fusion, large pieces of chromosomes, orentire chromosomes, have been introduced. See European PatentApplication Nos. 773 288 and 843 961, the disclosures of which arehereby incorporated by reference. Additionally, KM™-mice, which are theresult of cross-breeding of Kirin's Tc mice with Medarex's minilocus(Humab) mice have been generated. These mice possess the HCtranschromosome of the Kirin mice and the kappa chain transgene of theMedarex mice (Ishida et al., Cloning Stem Cells, (2002) 4:91-102).

Human antibodies can also be derived by in vitro methods. Suitableexamples include, but are not limited to, phage display (ascommercialized by Cambridge Antibody Technology, Morphosys, Dyax,Biosite/Medarex, Xoma, Symphogen, Alexion (formerly Proliferon),Affimed) ribosome display (as commercialized by Cambridge AntibodyTechnology), yeast display, and the like.

Antibodies, as described herein, were prepared through the utilizationof the XENOMOUSE® technology, as described below. Such mice, then, arecapable of producing human immunoglobulin molecules and antibodies andare deficient in the production of murine immunoglobulin molecules andantibodies. Technologies utilized for achieving the same are disclosedin the patents, applications, and references disclosed in the backgroundsection herein. In particular, however, a preferred embodiment oftransgenic production of mice and antibodies therefrom is disclosed inU.S. patent application Ser. No. 08/759,620, filed Dec. 3, 1996 andInternational Patent Application Nos. WO 98/24893, published Jun. 11,1998 and WO 00/76310, published Dec. 21, 2000, the disclosures of whichare hereby incorporated by reference. See also Mendez et al. NatureGenetics 15:146-156 (1997), the disclosure of which is herebyincorporated by reference.

Through the use of such technology, fully human monoclonal antibodies toa variety of antigens have been produced. Essentially, XENOMOUSE® linesof mice are immunized with an antigen of interest (e.g. HER-3),lymphatic cells (such as B-cells) are recovered from the mice thatexpressed antibodies, and the recovered cell lines are fused with amyeloid-type cell line to prepare immortal hybridoma cell lines. Thesehybridoma cell lines are screened and selected to identify hybridomacell lines that produced antibodies specific to the antigen of interest.Provided herein are methods for the production of multiple hybridomacell lines that produce antibodies specific to HER-3. Further, providedherein are characterization of the antibodies produced by such celllines, including nucleotide and amino acid sequence analyses of theheavy and light chains of such antibodies.

In general, antibodies produced by the fused hybridomas were human IgG1heavy chains with fully human kappa light chains. Antibodies describedherein possess human IgG4 heavy chains as well as IgG1 heavy chains.Antibodies can also be of other human isotypes, including IgG2 or IgG3.The antibodies possessed high affinities, typically possessing a K_(D)of from about 10⁻⁶ through about 10⁻¹³ M or below, when measured bysolid phase and cell-based techniques.

Another aspect of the present invention relates to an isolated nucleicacid molecule encoding a binding protein of the invention. Within thecontext of the present invention, the term “isolated nucleic acidmolecule”, as used herein, means a polynucleotide of genomic, cDNA, orsynthetic origin or some combination thereof, which by virtue of itsorigin, the “isolated nucleic acid molecule” (1) is not associated withall or a portion of a polynucleotide in which the “isolatedpolynucleotide” is found in nature, (2) is operably linked to apolynucleotide which it is not linked to in nature, or (3) does notoccur in nature as part of a larger sequence. Further, the term “nucleicacid molecule”, as referred to herein, means a polymeric form ofnucleotides of at least 10 bases in length, either ribonucleotides ordeoxynucleotides or a modified form of either type of nucleotide, suchas nucleotides with modified or substituted sugar groups and the like.The term also includes single and double stranded forms of DNA.

In a one embodiment of the present invention, a nucleic acid molecule ofthe invention is operably linked to a control sequence. The term“control sequence”, as used herein, refers to polynucleotide sequencesthat are necessary to effect the expression and processing of codingsequences to which they are ligated. The nature of such controlsequences differs depending upon the host organism. In prokaryotes, suchcontrol sequences generally include promoters, ribosomal binding sites,and transcription termination sequences. In eukaryotes, generally, suchcontrol sequences include promoters and transcription terminationsequences. In accordance with the present invention, the term “controlsequence” is intended to include, at a minimum, all components whosepresence is essential for expression and processing, and can alsoinclude additional components whose presence is advantageous, forexample, leader sequences and fusion partner sequences. Furthermore, theterm “operably linked”, as used herein, refers to positions ofcomponents so described which are in a relationship permitting them tofunction in their intended manner. Moreover, according to the presentinvention, an expression control sequence operably linked to a codingsequence is ligated in such a way that expression of the coding sequenceis achieved under conditions compatible with the expression controlsequence.

A further aspect of the present invention is a vector comprising anucleic acid molecule that encodes a binding protein of the invention.The nucleic acid molecule can be operably linked to a control sequence.Furthermore, the vector may additionally contain a replication origin ora selection marker gene. Examples of vectors that may be used inaccordance with the present invention are e.g. plasmids, cosmids,phages, viruses, etc.

Another aspect of the present invention relates to a host celltransformed with a nucleic acid molecule or vector of the invention.Transformation could be done by any known method for introducingpolynucleotides into a host cell, including for example packaging thepolynucleotide in a virus (or into a viral vector) and transducing ahost cell with the virus (or vector) or by transfection procedures knownin the art, as exemplified by U.S. Pat. Nos. 4,399,216, 4,912,040,4,740,461, and 4,959,455, which patents are hereby incorporated hereinby reference. Particularly, methods for introducing heterologouspolynucleotides into mammalian cells are well known in the art andinclude dextran-mediated transfection, calcium phosphate precipitation,polybrene mediated transfection, protoplast fusion, electroporation,encapsulation of the polynucleotide(s) in liposomes, and directmicroinjection of the DNA into nuclei. Examples of host cells that maybe used according to the present invention are hybridomas eukaryoticcells such as mammalian cells, e.g. hamster, rabbit, rat, pig, mouse orother animal cells; plant cells and fungal cells, e.g. corm, tobacco,Saccharomyces cerevisiae, Pichia pastoris; prokaryotic cells such as E.coli; and other cells used in the art for the production of antibodies.Especially mammalian cell lines available as hosts for expression arewell known in the art and include many immortalized cell lines availablefrom the American Type Culture Collection (ATCC), including but notlimited to Chinese hamster ovary (CHO) cells, HeLa cells, baby hamsterkidney (BHK) cells, monkey kidney cells (COS), human hepatocellularcarcinoma cells (e.g. Hep G2), and a number of other cell lines.

Yet another aspect of the present invention is a pharmaceuticalcomposition comprising as an active agent at least one binding proteinof the invention and pharmaceutically acceptable carriers, diluentsand/or adjuvants. The term “pharmaceutical composition”, as used herein,refers to a chemical compound or composition capable of inducing adesired therapeutic effect when properly administered to a patient (TheMcGraw-Hill Dictionary of Chemical Terms, Parker, S., Ed., McGraw-Hill,San Francisco (1985), incorporated herein by reference). In accordancewith the present invention, the potency of the pharmaceuticalcomposition of the invention is based on the binding of the at least onebinding protein to HER-3. Preferably, this binding leads to a reductionof the HER-3-mediated signal transduction.

Furthermore, the term “carriers”, when used herein, includes carriers,excipients, or stabilizers that are nontoxic to the cell or mammal beingexposed thereto at the dosages and concentrations employed. Often thephysiologically acceptable carrier is an aqueous pH buffered solution ora liposome (a small vesicle composed of various types of lipids,phospholipids and/or surfactants which is useful for delivery of a drugto a mammal). Examples of physiologically acceptable carriers includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptides; proteins such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN™, polyethylene-glycol (PEG), and PLURONICS™.

In a one embodiment of the present invention, the at least one bindingprotein of the invention contained in the pharmaceutical composition iscoupled to an effector, such as calicheamicin, Auristatin-PE, aradioisotope or a toxic chemotherapeutic agent such as geldanamycin andmaytansine. In particular, these binding protein conjugates are usefulin targeting cells, e.g. cancer cells, expressing HER-3 for elimination.

Moreover, linking binding proteins of the invention to radioisotopese.g. provides advantages to tumor treatments. Unlike chemotherapy andother forms of cancer treatment, radioimmunotherapy or theadministration of a radioisotope-binding protein combination directlytargets the cancer cells with minimal damage to surrounding normal,healthy tissue. With this “magic bullet”, the patient can be treatedwith much smaller quantities of radioisotopes than other forms oftreatment available today. Preferred radioisotopes include yttrium⁹⁰(⁹⁰Y), indium¹¹¹ (¹¹¹In), ¹³¹I, ⁹⁹mTc, radiosilver-111, radiosilver-199,and Bismuth²¹³. The linkage of radioisotopes to binding proteins of theinvention may e.g. be performed with conventional bifunctional chelates.Since silver is monovalent, for radiosilver-111 and radiosilver-199linkage, sulphur-based linkers may be used (Hazra et al., Cell Biophys.24-25, 1-7 (1994)). Linkage of silver radioisotopes may involve reducingthe immunoglobulin with ascorbic acid. Furthermore, tiuxetan is anMX-DTPA linker chelator attached to ibritumomab to form ibritumomabtiuxetan (Zevalin) (Witzig, T. E, Cancer Chemother. Pharmacol. 48 Suppl1, 91-5 (2001). Ibritumomab tiuxetan can react with radioisotypes suchas indium¹¹¹ (¹¹¹In) or ⁹⁰Y to form ¹¹¹In-ibritumomab tiuxetan and⁹⁰Y-ibritumomab tiuxetan, respectively.

Furthermore, a binding protein of the invention, particularly when usedto treat cancer, may be conjugated with toxic chemotherapeutic drugssuch as calicheamicin (Hamann et al., Bioconjug. Chem. 13(1), 40-6(2002), geldanamycin (Mandler et al., J. Natl. Cancer Inst., 92(19),1549-51 (2000)) and maytansine, for example, the maytansinoid drug, DM1(Liu et al., Proc. Natl. Acad. Sci. U.S.A. 93:8618-8623 (1996)).Different linkers that release the drugs under acidic or reducingconditions or upon exposure to specific proteases may be employed withthis technology. According to the present invention, a binding proteinof the invention may be conjugated as described in the art.

Auristatin-PE, e.g. is an antimicrotubule agent that is a structuralmodification of the marine, shell-less mollusk peptide constituentdolastatin 10. Auristatin-PE has both anti-tumor activity and anti-tumorvascular activity (Otani et al., Jpn. J. Cancer Res. 91(8), 837-44(2000)). For example, auristatin-PE inhibits cell growth and inducescell cycle arrest and apoptosis in pancreatic cancer cell lines (Li etal., Int. J. Mol. Med. 3(6), 647-53 (1999)). Accordingly, tospecifically target the anti-tumor activity and anti-tumor vascularactivities of auristatin-PE to particular tumors, auristatin-PE may beconjugated to the binding protein of the invention.

In a one embodiment of the present invention, the pharmaceuticalcomposition comprises at least one further active agent. Examples forfurther active agents, which may be used in accordance with the presentinvention, are antibodies or low molecular weight inhibitors of otherreceptor protein kinases, such as EGFR, HER-2, HER4, IGFR-1, or c-met,receptor ligands such as vascular endothelial factor (VEGF), cytotoxicagents, such as doxorubicin, cis-platin or carboplatin, cytokines orantineoplatic agents. Many antineoplastic agents are presently known inthe art. In one embodiment, the antineoplastic agent is selected fromthe group of therapeutic proteins including, but not limited to,antibodies or immunomodulatory proteins. In another embodiment theanti-neoplastic agent is selected from the group of small moleculeinhibitors or chemotherapeutic agents consisting of mitotic inhibitors,kinase inhibitors, alkylating agents, anti-metabolites, intercalatingantibiotics, growth factor inhibitors, cell cycle inhibitors, enzymes,topoisomerase inhibitors, histone deacetylase inhibitors, anti-survivalagents, biological response modifiers, anti-hormones, e.g.anti-androgens, and anti-angiogenesis agents. When the anti-neoplasticagent is radiation, treatment can be achieved either with an internal(brachytherapy BT) or external (external beam radiation therapy: EBRT)source.

The pharmaceutical composition of the present invention is especiallysuitable for the diagnosis, prevention or treatment of ahyperproliferative disease. The hyperproliferative disease may be, e.g.,associated with increased HER family signal transduction. Particularly,the disease can be associated with increased HER-3 phosphorylationand/or increased complex formation between HER-3 and other members ofthe HER family and/or increased PI₃ kinase activity and/or increasedc-jun terminal kinase activity and/or AKT activity and/or increased ERK2activity and/or PYK2 activity. Preferably, the hyperproliferativedisease is selected from the group consisting of breast cancer,gastrointestinal cancer, pancreatic cancer, prostate cancer, ovariancancer, stomach cancer, endometrial cancer, salivary gland cancer, lungcancer, kidney cancer, colon cancer, colorectal cancer, thyroid cancer,bladder cancer, glioma, melanoma or other HER-3 expressing oroverexpressing cancers, and the formation of tumor metastases.

In accordance with the present invention, the term “prevention ortreatment”, when used herein, refers to both therapeutic treatment andprophylactic or preventative measures, wherein the patient in need is toprevent or slow down (lessen) the targeted pathologic condition ordisorder. Those in need of prevention or treatment include those alreadywith the disorder as well as those prone to have the disorder or thosein whom the disorder is to be prevented. The patient in need ofprevention or treatment is a mammalian patient, i.e. any animalclassified as a mammal, including humans, domestic and farm animals, andzoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep,pigs, goats, rabbits, etc. Preferably, the patient in need of treatmentis a human patient.

According to the present invention, the pharmaceutical composition ofthe invention may be formulated by mixing the active agent(s) withphysiologically acceptable carriers, diluents and/or adjuvants, andoptionally other agents that are usually incorporated into formulationsto provide improved transfer, delivery, tolerance, and the like. Thepharmaceutical composition of the invention may be formulated e.g. inthe form of lyophilized formulations, aqueous solutions, dispersions orsolid preparations, such as tablets, dragees or capsules. A multitude ofappropriate formulations can be found in the formulary known to allpharmaceutical chemists: Remington's Pharmaceutical Sciences (18^(th)ed, Mack Publishing Company, Easton, Pa. (1990)), particularly Chapter87 by Block, Lawrence, therein. These formulations include, for example,powders, pastes, ointments, jellies, waxes, oils, lipids, lipid(cationic or anionic) containing vesicles (such as Lipofectin™), DNAconjugates, anhydrous absorption pastes, oil-in-water and water-in-oilemulsions, emulsions carbowax (polyethylene glycols of various molecularweights), semi-solid gels, and semi-solid mixtures containing carbowax.Any of the foregoing mixtures may be appropriate in treatments andtherapies in accordance with the present invention, provided that theactive agent in the formulation is not inactivated by the formulationand the formulation is physiologically compatible and tolerable with theroute of administration. See also Baldrick P., “Pharmaceutical excipientdevelopment: the need for preclinical guidance.”, Regul. Toxicol.Pharmacol. 32(2), 210-218 (2000); Wang W., “Lyophilization anddevelopment of solid protein pharmaceuticals.”, Int. J. Pharm. 203(1-2),1-60 (2000); Charman W. N., “Lipids, lipophilic drugs, and oral drugdelivery-some emerging concepts.”, J. Pharm. Sci. 89(8), 967-978 (2000);Powell et al., “Compendium of excipients for parenteral formulations”,PDA J. Pharm. Sci. Technol. 52, 238-311 (1998); and the citationstherein for additional information related to formulations, excipientsand carriers well known to pharmaceutical chemists.

Another aspect of the present invention pertains to the use of at leastone isolated binding protein of the invention, and optionally at leastone other active agent, e.g. at least one anti-neoplastic agent asdescribed above, in admixture with pharmaceutically acceptable carriers,diluents and/or adjuvants, for the manufacture of a pharmaceuticalcomposition for the diagnosis, prevention or treatment of ahyperproliferative disease. Preferably, the pharmaceutical compositionis a pharmaceutical composition as described above and thehyperproliferative disease is a hyperproliferative disease as mentionedabove.

Yet another aspect of the present invention is concerned with a methodfor diagnosing diseases or conditions associated with the expression ofHER-3, comprising contacting a sample with a binding protein of theinvention, and detecting the presence of HER-3 in the sample. The samplemay be a cell that shows expression of HER-3, such as a tumor cell, ablood sample or another suitable sample. In a preferred embodiment ofthe present invention, the diseases or conditions associated with theexpression of HER-3 are the hyperproliferative diseases defined above.

According to the present invention, the method may, e.g., be used forthe detection of HER-3 antigen in a cell, for the determination of HER-3antigen concentration in patients suffering from a hyperproliferativedisease as mentioned above or for the staging of said hyperproliferativedisease in a patient. In order to stage the progression of ahyperproliferative disease in a subject under study, or to characterizethe response of the subject to a course of therapy, a sample of bloodcan, e.g., be taken from the subject and the concentration of the HER-3antigen present in the sample is determined. The concentration soobtained is used to identify in which range of concentrations the valuefalls. The range so identified correlates with a stage of progression ora stage of therapy identified in the various populations of diagnosedsubjects, thereby providing a stage in the subject under study. A biopsyof the disease, e.g. cancer, tissue obtained from the patient may alsobe used assess the amount of HER-3 antigen present. The amount of HER-3antigen present in the disease tissue may be assessed byimmunohistochemistry, ELISA or antibody arrays using HER3 antibodies ofthe invention. Other parameters of diagnostic interest are thedimerization state as well as the dimerization partners of the HER3protein and the activation state of it and its partners. Proteinanalytical methods to determine those parameters are well known in theart and are among others western blot and immunoprecipitationtechniques, FACS analysis, chemical crosslinking, bioluminescenceresonance energy transfer (BRET), fluorescence resonance energy transfer(FRET) and the like (e.g. Price et al, Methods in Molecular Biology,218: 255-268 (2002) or the eTag technology (WO0503707, WO04091384,WO04011900).

Furthermore, the present invention relates in another aspect to a methodfor preventing or treating diseases or conditions associated with theexpression of HER-3 in a patient, comprising administering to a patientin need thereof an effective amount of at least one binding protein ofthe invention. Preferably, the diseases or conditions associated withthe expression of HER-3 are the hyperproliferative diseases definedabove. The patient in need of prevention or treatment is a mammalianpatient, i.e. any animal classified as a mammal, including humans,domestic and farm animals, and zoo, sports, or pet animals, such asdogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc.Preferably, the patient in need is a human patient.

In a preferred embodiment of the present invention, the method forpreventing or treating a hyperproliferative disease in a patient in needthereof comprises administering to the patent an effective amount of atleast one binding protein of the invention and additionally at least oneother active agent, e.g., at least one antineoplastic agent as mentionedabove. Preferably, the method is for inhibiting abnormal cell growth,migration or invasion.

In addition to classical modes of administration of potential bindingprotein therapeutics, e.g. via the above mentioned formulations, newlydeveloped modalities of administration may also be useful according tothe present invention. For example, local administration of ¹³¹I-labeledmonoclonal antibody for treatment of primary brain tumors after surgicalresection has been reported. Additionally, direct stereotacticintracerebral injection of monoclonal antibodies and their fragments isalso being studied clinically and pre-clinically. Intracarotidhyperosmolar perfusion is an experimental strategy to target primarybrain malignancy with drug conjugated human monoclonal antibodies.

Depending on the type and severity of the condition to be treated, about1 μg/kg to 15 mg/kg of the at least one binding protein of the inventionmay be administered to a patient in need thereof, e.g. by one or moreseparate administrations or by continuous infusion. A typical dailydosage might range from about 1 μg/kg to about 100 mg/kg or more,depending on the factors mentioned above. For repeated administrationsover several days or longer, depending on the condition to be treated,the treatment is sustained until a desired suppression of diseasesymptoms occurs.

The dose of the at least one antineoplastic agent administered dependson a variety of factors. These are, for example, the nature of theagent, the tumor type or the route of administration. It should beemphasized that the present invention is not limited to any dose.

Finally, the present invention relates in a further aspect to a kit forthe diagnosis, prevention or treatment of hyperproliferative diseasesassociated with HER-3 mediated signal transduction, comprising the atleast one binding protein and/or nucleic acid molecule and/or vector ofthe invention. In addition, the kit of the invention can furthercomprise at least one other active agent, e.g. at least one otherantineoplastic agent as mentioned above.

Further, the present invention shall be explained by the followingExamples and the accompanying drawing figures.

EXAMPLES

The following examples, including the experiments conducted and resultsachieved, are provided for illustrative purposes only and are not to beconstrued as limiting upon the present invention.

Example 1 HER-3 Antigen and Cell Line Preparation

In the present study, recombinant HER-3 proteins were prepared. Theextracellular domain of HER-3 (ECD) cDNA was cloned by polymerase chainreaction (PCR) from pcDNA3-HER-3 (expression vector with full lengthhuman HER-3, C. Wallasch et al., EMBO J. 14, 4267-4275) with primersbased on the sequence of HER-3 (Genebank AccNr. NM_(—)001982; SEQ IDNO:390).

The primers used for the amplification of HER-3 were as follows:

Forward primer: (SEQ ID NO: 233) 5′-CGGGATCCATGTCCTAGCCTAGGGGC-3′Reverse primer: (SEQ ID NO: 234)5′-GCTCTAGATTAATGATGATGATGATGATGTTGTCCTAAAC AGTCTTG-3′

The PCR product was digested with BamH1 and XbaI and ligated into pcDNA3(Invitrogen) digested with BamH1 and XbaI. Plasmids were transfectedinto HEK293 cells using a CaPO₄ method. The HER-3-HIS fusion protein waspurified from harvested conditioned media via Ni-NTA affinitychromatography.

RatI HER-3 cells were generated by retroviral gene transfer. Briefly,GP+E 86 cells (3×10⁵) were seeded on a 60 mm culture disc andtransfected with 2 μg/ml pIXSN vector or pIXSN-HER-3 cDNA (C. Wallasch,PhD Thesis, Max-Planck Institute of Biochemistry, Martinsried, Germany)using the calcium phosphate method. After 24 h medium was replaced byfresh medium and the GP+E 86 cells were incubated for 4-8 hrs.Subconfluent Rat1 cells (2×10⁵ cells per 6 cm dish) were then incubatedwith supernatants of GP+E 86 cells releasing high titer pLXSN orpLXSN-HER-3, p virus (>1×106 G418 c.f.u./ml; m.o.i. of 10) for 4-12 h inthe presence of Polybrene (4 mg/ml; Aldrich). After changing the medium,selection of RatI cells with G418 was started. Usually, stable cloneswere picked after selection for 21 days.

Example 2 HER-3 Expression in Human Cancer Cell Lines

Receptor tyrosine kinases, as for example HER-3, play a crucial role inthe initiation and progression of hyperproliferative diseases such asthe transition from benign hyperplastic cell growth towards a malignantcarcinoma. Since HER-3 expression varies between tumor cells and normaltissue an analysis of HER-3 expression is a critical factor foridentification of patient subgroups that would benefit from treatmentwith binding proteins of the invention. Thus, HER-3 expression wasquantified in a panel of human cancer cell lines to elucidate the roleof HER-3 in human cancer formation. Cancer cell lines were grown asrecommended by the ATCC. In detail, 10⁵ cells were harvested with 10 mMEDTA in PBS, washed once with FACS buffer (PBS, 3% FCS, 0.4% azide) andseeded on a 96-well round bottom plate. The cells were spun for 3 min at1000 rpm to remove supernatant and then resuspended with α-HER-3antibody 2D1D12 (WO03013602) (3 μg/ml). Cell suspensions were incubatedon ice for 1 hr, washed twice with FACS buffer and resuspended withsecondary antibody (100 μl/well) donkey-anti-human-PE (Jackson) diluted1:50 in FACS buffer. The cell suspensions were incubated on ice and inthe dark for 30 min, washed twice with FACS buffer and analyzed (FACS,Beckman Coulter). FIG. 1 shows representative results of the analysisand demonstrates that HER-3 is expressed in a variety of human cancers.

Example 3 Immunization and Tittering

The HER-3 ECD protein, that was prepared as described in Example 1 andC32 cells (Human melanoma; ATCC #CRL-1585) were used as an antigen.Monoclonal antibodies against HER-3 were developed by sequentiallyimmunizing XenoMouse® mice (XenoMouse® strains: XMG1 and XMG4, Abgenix,Inc. Fremont, Calif.). XenoMouse® animals were immunized via footpadroute for all injections. The total volume of each injection was 50 μlper mouse, 25 μl per footpad.

For cohort #1 (10 XMG1 mice), the initial immunization was with 10 μg ofHER-3 ECD protein admixed 1:1 (v/v) with TITERMAX GOLD® (Sigma,Oakville, ON) per mouse. The subsequent five boosts were made with 10 μgof HER-3 ECD protein admixed 1:1 (v/v) with 100 μg alum gel (Sigma,Oakville, ON) in pyrogen-free D-PBS. The sixth boost consisted of 10 μgof HER-3 ECD protein admixed 1:1 (v/v) with TITERMAX GOLD®. The seventhinjection consisted of 10 μg of HER-3 ECD protein admixed 1:1 v/v with100 μg alum gel. A final boost was made with 10 μg HER-3 ECD protein inpyrogen-free DPBS, without adjuvant. The XenoMouse® mice were immunizedon days 0, 4, 7, 11, 15, 20, 24, and 29 for this protocol and fusionswere performed on day 33. The two bleeds were made through Retro-OrbitalBleed procedure on day 13 after the fourth boost, on day 19 after thesixth boost. There was no cohort #2.

For Cohort #3 (10 XMG1 mice) and Cohort #4 (10 XMG4 mice), the first 15injection was with 10⁷ C32 cells in pyrogen-free Dulbecco's PBS (DPBS)admixed 1:1 (v/v) with TITERMAX GOLD® per mouse. The next four boostswere with 10⁷ C32 cells in pyrogen-free DPBS, admixed with 25 μg ofAdju-Phos and 10 μg CpG per mouse. The sixth boost was with 10⁷ C32cells in pyrogen-free DPBS, admixed 1:1 (v/v) with TITERMAX GOLD® permouse. The seventh, eighth, ninth boosts were with 107 C32 cells inpyrogen-free DPBS, admixed with 25 μg of Adju-Phos and 10 μg CpG permouse. From tenth to fourteen boosts were 5 μg of HER-3 ECD protein inpyrogen-free DPBS, admixed with 25 μg of Adju-Phos and 10 μg CpG permouse. A final boost consisted of 5 μg of HER-3 ECD protein inpyrogen-free DPBS, without adjuvant. Both Cohort #3 and #4, theXenoMouse® mice were immunized on days 0, 3, 7, 11, 14, 17, 21, 24, 28,33, 35, 38, 42 and 45 for this protocol and fusions were performed onday 49. The three bleeds were made through Retro-Orbital Bleed procedureon day 12 after the fourth boost, on day 19 after the sixth boost and onday 40 after twelfth boost.

Selection of Animals for Harvest by Titer

For cohort #1, anti-HER-3 antibody titers in the serum from immunizedXenoMouse® mice were determined by ELISA against HER-3 ECD protein. Thespecific titer of each XenoMouse® animal was determined from the opticaldensity at 650 nm and is shown in Table 1 below. The titer value is thereciprocal of the greatest dilution of sera with an OD reading two-foldthat of background. Therefore, the higher the number, the greater wasthe humoral immune response to HER-3 ECD.

TABLE 1 Cohort #1, XMG1 Mouse ID After 4 inj. After 6 inj. P3421 8,00011,000 P3422 850 2,600 P3423 2,700 5,200 P3424 3,200 9,100 P3425 5,4002,500 P3426 700 1,500 P3427 5,800 7,000 P3428 3,900 4,300 P3429 2,2002,500 P34210 600 850 NC 250 175 PC 377,000 311,000 NC mAb IL-8, D39.2.1PC xHER-3-2D1D12

For cohort #3 and #4, anti-HER-3 antibody titers in the serum fromimmunized XenoMouse® mice were determined by FACS using Rat1/HER-3 cells(antigen positive cell line) cells and Rat1/pLSXN cells (antigennegative cell line). Data are presented as geometric mean (GeoMean)fluorescent intensity of cell anti-HER-3 cell staining by serialdilutions of serum samples.

TABLE 2 Cohort #3, XMG1 After 6 inj. After 12 inj. Sample pos cells negcells pos cells neg cells Mouse ID dilution GeoMean GeoMean GeoMeanGeoMean Q832-1 1:50 9 10 11 10 1:250 6 9 6 6 1:1250 6 7 4 4 Q832-2 1:508 10 29 42 1:250 7 8 11 11 1:1250 5 6 6 5 Q832-3 1:50 7 12 11 9 1:250 57 5 5 1:1250 5 5 4 4 Q832-4 1:50 6 10 9 9 1:250 6 6 5 5 1:1250 5 5 4 4Q832-5 1:50 11 11 17 13 1:250 10 9 7 6 1:1250 6 8 5 4 Q832-6 1:50 7 1115 14 1:250 7 7 7 6 1:1250 5 6 6 4 Q832-7 1:50 8 11 7 15 1:250 6 7 5 51:1250 5 5 4 4 Q832-8 1:50 7 8 11 20 1:250 6 6 7 8 1:1250 5 5 5 4 Q832-91:50 7 12 15 16 1:250 6 8 6 5 1:1250 6 6 4 4 Q832-10 1:50 8 13 34 381:250 6 8 9 8 1:1250 6 6 5 4

TABLE 3 Cohort #4, XMG4 After 6 inj. After 12 inj. Mouse Sample poscells neg cells pos cells neg cells ID dilution GeoMean GeoMean GeoMeanGeoMean Q856-1 1:50 4 6 91 44 1:250 4 5 32 18 1:1250 4 4 19 10 Q856-21:50 4 8 148 54 1:250 4 5 89 23 1:1250 4 4 42 9 Q856-3 1:50 4 5 72 141:250 4 4 28 6 1:1250 4 4 18 4 Q856-4 1:50 4 5 11 49 1:250 4 5 10 171:1250 4 4 8 7 Q856-5 1:50 4 4 74 20 1:250 4 4 30 14 1:1250 4 4 16 6Q856-6 1:50 4 5 86 21 1:250 4 4 32 10 1:1250 4 4 16 5 Q856-7 1:50 5 6 7432 1:250 4 5 32 14 1:1250 4 4 16 6 Q856-8 1:50 4 5 106 14 1:250 4 4 45 61:1250 4 4 22 4 Q856-9 1:50 5 6 53 22 1:250 4 4 17 11 1:1250 4 4 11 5Q856-10 1:50 4 5 72 53 1:250 4 4 26 17 1:1250 4 4 15 7

Example 4 Recovery of Lymphocytes, B-Cell Isolations, Fusions andGeneration of Hybridomas

Immunized mice were sacrificed and the lymph nodes were harvested andpooled from each cohort. The lymphoid cells were dissociated by grindingin DMEM to release the cells from the tissues, and the cells weresuspended in DMEM. The cells were counted, and 0.9 ml DMEM per 100million lymphocytes was added to the cell pellet to resuspend the cellsgently but completely. Using 100 μl of CD90+ magnetic beads per 100million cells, the cells were labeled by incubating the cells with themagnetic beads at 4° C. for 15 min. The magnetically-labeled cellsuspension containing up to 10⁸, positive cells (or up to 2×10⁹ totalcells) was loaded onto a LS+ column and the column washed with DMEM. Thetotal effluent was collected as the CD90-negative fraction (most ofthese cells were expected to be B cells).

The fusion was performed by mixing washed enriched B cells from aboveand nonsecretory myeloma P3X63Ag8.653 cells purchased from ATCC (Cat.No. CRL 1580) (Kearney et al, J. Immunol. 123, 1979, 1548-1550) at aratio of 1:1. The cell mixture was gently pelleted by centrifugation at800 g. After complete removal of the supernatant, the cells were treatedwith 2 to 4 ml of pronase solution (CalBiochem, Cat. No. 53702; 0.5mg/ml in PBS) for no more than 2 min. Then 3 to 5 ml of FBS was added tostop the enzyme activity and the suspension was adjusted to 40 ml totalvolume using electro cell fusion solution, ECFS (0.3 M sucrose, Sigma,Cat. No. S7903, 0.1 mM magnesium acetate, Sigma, Cat. No. M2545, 0.1 mMcalcium acetate, Sigma, Cat. No. C4705). The supernatant was removedafter centrifugation and the cells were resuspended in 40 ml ECFS. Thiswash step was repeated and the cells again were resuspended in ECFS to aconcentration of 2×10⁶ cells/ml.

Electro-cell fusion was performed using a fusion generator, modelECM2001, Genetronic, Inc., San Diego, Calif. The fusion chamber sizeused was 2.0 ml, using the following instrument settings: Alignmentcondition: voltage: 50 V, time: 50 sec; membrane breaking at: voltage:3000 V, time: 30 μsec; post-fusion holding time: 3 sec.

After ECF, the cell suspensions were carefully removed from the fusionchamber under sterile conditions and transferred into a sterile tubecontaining the same volume of Hybridoma Culture Medium (DMEM (JRHBiosciences), 15% FBS (Hyclone), supplemented with L-glutamine,pen/strep, OPI (oxaloacetate, pyruvate, bovine insulin) (all from Sigma)and IL-6 (Boehringer Mannheim). The cells were incubated for 15 to 30min at 37° C., and then centrifuged at 400 g for five min. The cellswere gently resuspended in a small volume of Hybridoma Selection Medium(Hybridoma Culture Medium supplemented with 0.5×HA (Sigma, Cat. No.A9666)), and the volume was adjusted appropriately with more HybridomaSelection Medium, based on a final plating of 5×10⁶ B cells total per96-well plate and 200 μl per well. The cells were mixed gently andpipetted into 96-well plates and allowed to grow. On day 7 or 10,one-half the medium was removed, and the cells were re-fed withHybridoma Selection Medium.

Example 5 Selection of Candidate Antibodies by ELISA

After 14 days of culture, primary screening of hybridoma supernatantsfrom the cohort #1 (mice in cohort one were split arbitrarily intofusion #1 and #2) for HER-3-specific antibodies was performed by ELISAusing purified his-tagged HER-3 ECD and counter-screening against anirrelevant his-tagged protein by ELISA using goat anti-huIgGFc-HRP(Caltag Inc., Cat. No. H10507, using concentration was 1:2000 dilution)to detect human IgG binding to HER-3 ECD immobilized on ELISA plates.The old culture supernatants from the positive hybridoma cells growthwells based on primary screen were removed and the HER-3 positivehybridoma cells were suspended with fresh hybridoma culture medium andwere transferred to 24-well plates. After 2 days in culture, thesesupernatants were ready for a secondary confirmation screen. In thesecondary confirmation screen for HER-3 specific fully human IgGkantibodies, the positives in the first screening were screened by ELISAwith two sets of detective antibodies: goat anti-huIgGFc-HRP (CaltagInc., Cat. No. H10507, using concentration was 1:2000 dilution) forhuman gamma chain detection and goat anti-hIg kappa-HRP (SouthernBiotechnology, Cat. No. 2060-05) for human kappa light chain detection.There were 91 fully human IgG/kappa HER-3 specific monoclonal antibodiesthat were generated from cohort #1.

Example 6 Selection of Candidate Antibodies by FMAT/FACS

After 14 days of culture, hybridoma supernatants from the cohort #3 and#4 (fusion #3 and #4) were screened for HER-3-specific monoclonalantibodies by FMAT. In the primary screen, hybridoma supernatants at1:10 final dilution were incubated with Rat1-Her3 cells expressing humanHER-3 and 400 ng/ml Cy5-conjugated Goat F(ab′)2 anti-human IgG,Fc-specific antibody (Jackson ImmunoResearch, Cat. No. 109-176-098) atroom temperature for 6 hr. The binding of antibodies and detectionantibodies complex to cells were measured by FMAT (Applied Biosystems).Non-specific binding of antibodies to the cells was determined by theirbinding to parental Rat1 cells. A total of 420 hybridomas producingHER-3-specific antibodies were selected from primary screen of fusion#3. The supernatants from these expanded cultures were tested againusing the same FMAT protocol and 262 of them were confirmed to bind toHER-3 expressing cells specifically. A total of 193 hybridomas producingHER-3 specific antibodies were selected from primary screen of fusion#4. The supernatants from these expanded cultures were tested by FACSand 138 of them were confirmed to bind to HER-3 expressing cellsspecifically. In the FACS confirmation assay, Rat1-Xher3 cells andparental Rat1 cells (as negative control) were incubated with hybridomasupernatants at 1:2 dilution for 1 hr at 40 C in PBS containing 2% FBS.Following washing with PBS, the binding of antibodies to the cells weredetected by 2.5 μg/ml Cy5-conjugated Goat F(ab′)2 anti-human IgG,Fc-specific antibody (JIR#109-176-098) and 5 μg/ml PE-conjugated GoatF(ab′)2 anti-human kappa-specific antibody (SB#2063-09). After removingthe unbound antibodies by washing with PBS, the cells were fixed bycytofix (BD#51-2090KZ) at 1:4 dilution and analyzed by FACSCalibur.

Example 7 Selection of Hybridomas for Cloning

Antibodies from cohorts 1 and 2 were selected for hybridoma cloningbased on specificity for HER-3 over HER1 (EGFR), HER-2 and HER-4 inELISA using purified recombinant extra-cellular domains available from,for example R&D Biosystems, and FACS-based analysis of human tumor celllines expressing different HER family members, and a >5-time increase inmean fluorescent intensity in FACS staining for HER-3 positive cellsover background. Based on these criteria, a total of 23 hybridoma lineswere selected for cloning by limiting dilution cell plating.

Antibodies from cohorts 3 and 4 were selected for hybridoma cloningbased on specificity for HER-3 over HER-1 (EGFR), HER-2 and HER-4 plusthree other criteria. The first criterion was an ELISA screen forantibodies with epitopes contained within the L2 domain of HER-3 (seeExample “Structural Analysis of anti-HER-3 Antibodies in the Invention).

The second criterion was neutralization of binding of biotinylatedheregulin-alpha to HER-3 expressing cells in a FACS based assay. SKBR-3cells were harvested, washed in culture medium, pelleted viacentrifugation and resuspended in culture medium. Resuspended cells werealiquoted into 96-well plates. The plates were centrifuged to pellet thecells. Test antibodies in exhaust hybridoma supernatants were added at25 μl/well and incubated for 1 hr on ice to allow antibody binding.Fifty μl of a 10 nM heregulin-alpha (R&D Biosystems, Minneapolis, Minn.)solution was added to each well for a final concentration of 5 nM andincubated on ice for 1.5 hr. Cells were washed in 150 μl PBS, pelletedby centrifugation and the supernatant removed. Cells were resuspended in50 μl of goat anti-HRG-alpha polyclonal antibody at 10 μg/ml andincubated for 45 min of ice. Cells were washed in 200 μl PBS, pelletedby centrifugation and the supernatant removed. Fifty μl of a solution ofrabbit Cy5-labeled anti-goat polyclonal antibody at 5 μg/ml plus 7AAD at10 μg/ml was added and incubated on ice for 15 min. Cells were washed in200 μl PBS, pelleted by centrifugation and the supernatant removed. Thecells were resuspended in 100 μl of FACS buffer and read in the FACS.Test HER-3 antibodies that reduced binding of heregulin-alpha were thosethat had lowest fluorescence intensity. As positive controls, 1:5 serialdilutions from 10,000 ng/ml to 16 ng/ml of a mouse HER-3 mAb (105.5) orthe human IgG1 HER-3 mAb, U1-49 was used. Negative controls wereheregulin-alpha alone, cells alone, goat anti-heregulin-alpha polyclonalantibody alone and rabbit Cy5-labeled anti-goat polyclonal antibodyalone.

The third criterion was relative ranking for affinity and/or higherrelative mean fluorescence intensity in FACS using HER-3 expressing celllines. Relative ranking for affinity was performed by normalizingHER-3-specific antibody concentrations and plotting versus data fromlimiting antigen ELISA as follows.

Normalization of Antigen Specific Antibody Concentrations Using HighAntigen ELISA

Using an ELISA method, supernatants for concentration of antigenspecific antibody were normalized. Using two anti-HER-3 human IgG1antibodies from cohort 1 of known concentration titrated in parallel, astandard curve was generated and the amount of antigen specific antibodyin the test hybridoma supernatants from cohorts 3 and 4 were compared tothe standard. In this way, the concentration of human HER3 IgG antibodyin each hybridoma culture was estimated.

Neutravidin plates were made by coating neutravidin @ 8 μg/ml in1×PBS/0.05% sodium azide on Costar 3368 medium binding plates at 50ul/well with overnight incubation at 4° C. The next day the plates wereblocked with 1×PBS/1% skim milk. Photobiotinylated his-tagged-HER-3 ECD@ 500 ng/ml in 1×PBS/1% skim milk was bound to the neutravidin plates byincubating for 1 hour at room temperature. Hybridoma supernatant,serially diluted 1:2.5 from a starting dilution of 1:31 to a finaldilution of 1:7568 in 1×PBS/1% skim milk/0.05% azide, was added at 50μl/well, and then incubated for 20 hours at room temperature. Seriallydilutions were used to ensure obtaining OD readings for each unknown inthe linear range of the assay. Next, a secondary detection antibody,goat anti human IgG Fc HRP at 400 ng/ml in 1×PBX/1% skim milk was addedat 50 ul/well. After 1 hour at room temperature, the plates were againwashed 5 times with water and 50 μL of one-component TMB substrate wereadded to each well. The reaction was stopped after 30 minutes by theaddition of 50 μl of 1M hydrochloric acid to each well and the plateswere read at wavelength 450 nm. A standard curve was generated from thetwo IgG1 HER-3 mAbs from cohort 1, serially diluted at 1:2 from 1000ng/ml to 0.06 ng/ml and assessed in ELISA using the above protocol. Foreach unknown, OD readings in the linear range of the assay were used toestimate the concentration of human HER-3 IgG in each sample.

The limited antigen analysis is a method that affinity ranks theantigen-specific antibodies prepared in B-cell culture supernatantsrelative to all other antigen-specific antibodies. In the presence of avery low coating of antigen, only the highest affinity antibodies shouldbe able to bind to any detectable level at equilibrium. (See, e.g., PCTPublication WO/03048730A2 entitled “IDENTIFICATION OF HIGH AFFINITYMOLECULES BY LIMITED DILUTION SCREENING” published on Jun. 12, 2003). Inthis instance, two mAbs from cohort 1, both of known concentration andknown KD, were used as benchmarks in the assay.

Neutravidin plates were made by coating neutravidin at 8 μg/ml in1×PBS/0.05% sodium azide on Costar 3368 medium binding plates at 50ul/well with overnight incubation at 4° C. The next day the plates wereblocked with 1×PBS/1% skim milk. Biotinylated his-tagged-HER-3 ECD (50μl/well) was bound to 96-well neutravidin plates at five concentrations:125, 62.5, 31.2, 15.6, and 7.8 ng/ml in 1×PBS/1% skim milk for 1 hour atroom temperature. Each plate was washed 5 times with water. Hybridomasupernatants diluted 1:31 in 1×PBS/1% skim milk/0.05% azide were addedat 50 ul/well. After 20 hours incubation at room temperature on ashaker, the plates were again washed 5 times with dH₂O, Next, asecondary detection antibody, goat anti human IgG Fc HRP (Horsh RadishPeroxidase) at 400 ng/ml in 1×PBS/1% skim milk was added at 50 μl/well.After 1 hour at room temperature, the plates were again washed 5 timeswith dH₂O and 50 μL of one-component TMB substrate were added to eachwell. The reaction was stopped after 30 minutes by the addition of 50 μLof 1M hydrochloric acid to each well and the plates were read atwavelength 450 nm. OD readings from an antigen concentration thatyielded OD values in the linear range were used in for data analysis.

Plotting the high antigen data, which comparatively estimates specificantibody concentration (see above for details), versus the limitedantigen OD illustrated the relatively higher affinity antibodies, e.g.,those that bound had higher OD in the limited antigen assay while havinglower amounts of IgG HER-3 antibody in the supernatant.

Hybridomas from cohorts 3 and 4 for the 33 best performing antibodies inthese sets of assays were advanced to cloning by limiting dilutionhybridoma plating.

Alternatively, FACS analysis of HER-3 expression of RatI/pLXSN andRatI/HER-3 cells showed similar results (no crossreactivity withendogenous rat epitopes) (FIG. 2).

In detail 1×10⁵ cells were harvested with 10 mM EDTA in PBS, washed oncewith FACS buffer (PBS, 3% FCS, 0.4% azide) and seeded on a 96-well roundbottom plate. The cells were spun for 3 min at 1000 rpm to removesupernatant and then resuspended with the specific HER-family antibodies(3 μg/ml). Cell suspensions were incubated on ice for 45 min, washedtwice with FACS buffer and resuspended with secondary antibody (100μl/well) donkey-anti-human-PE (Jackson Immunoresearch, PA) diluted 1:50in FACS buffer. The cell suspensions were incubated on ice and in thedark for 30 min, washed twice with FACS buffer and analyzed (FACS,Beckman Coulter).

Example 8 Structural Analysis of Anti-HER-3 Antibodies of the Invention

In the following discussion, structural information related toantibodies prepared in accordance with the invention is provided. Inorder to analyze structures of antibodies produced in accordance withthe present invention, genes encoding the heavy and light chainfragments were amplified out of the particular hybridoma. Sequencing wasaccomplished as follows:

The VH and VL transcripts were amplified from individual hybridomaclones in 96 well plate using reverse transcriptase polymerase chainreaction (RT-PCR). Poly(A)+-mRNA was isolated from approximately 2×10⁵hybridoma cells using a Fast-Track kit (Invitrogen). Four PCR reactionswere run for each Hybridoma: two for light chain (kappa (κ), and two forgamma heavy chain (γ). The QIAGEN OneStep room temperature-PCR kit wasused for amplification (Qiagen, Catalog No. 210212). In the coupled roomtemperature-PCR reactions, cDNAs were synthesized with blend of roomtemperature enzymes (Omniscript and Sensiscript) using antisensesequence specific primer corresponded to C-κ, or to a consensus of theCH1 regions of Cγ genes. Reverse transcription was performed at 50° C.for 1 hr followed by PCR amplification of the cDNA by HotStarTaq DNAPolymerase for high specificity and sensitivity. Each PCR reaction useda mixture of 5′-sense primers; primer sequences were based on the leadersequences of VH and VK available at the Vbase website(http://vbase.mrc-cpe.cam.ac.uk/).

PCR reactions were run at 94° C. for 15 min, initial hot start followedby 40 cycles of 94° C. for 30 sec (denaturation), 60° C. for 30 sec(annealing) and 72° C. for 1 min (elongation).

PCR products were purified and directly sequenced using forward andreverse PCR primers using the ABI PRISM BigDye terminator cyclesequencing ready reaction Kit (Perkin Elmer). Both strands weresequenced using Prism dye-terminator sequencing kits and an ABI 377sequencing machine.

Sequence Analysis

Analyses of human V heavy and V kappa cDNA sequences of the HER3antibodies were accomplished by aligning the HER-3 sequences with humangermline V heavy and V kappa sequences using Abgenix in-house software(5AS). The software identified the usage of the V gene, the D gene andthe J gene as well as nucleotide insertions at the recombinationjunctions and somatic mutations. Amino acid sequences were alsogenerated in silico to identify somatic mutations. Similar results couldbe obtained with commercially available sequence analysis software andpublicly available information on the sequence of human V, D, and Jgenes, e.g., Vbase (http://vbase.mrc-cpe.cam.ac.uk/).

Molecular Cloning of mAb U1-59

Total RNA was extracted from the tissue culture well containing multiplehybridomas lineages, including the hybridoma lineage secreting antibodyU1-59. A heavy chain variable region was amplified using 5′-leader VHfamily specific primers, with 3′-C-gamma primer. A major band wasamplified using a VH4 primer, no other bands were visible. The VH4-34gamma fragment was cloned into pcDNA expression vector in frame with ahuman gamma 1 constant region gene.

An IgM heavy chain variable region was amplified using 5′ VH familyspecific primers with 3′ mu constant region primer. A major band wasamplified using VH2 primer, no other bands were visible. The VH2-5 mufragment was cloned into pCDNA expression vector in frame with a humanmu constant region gene. V kappa chains were amplified and sequenced.Four kappa chain RT-PCR products were identified. The products weresequenced and after sequence analysis via in silico translation, onlythree of them had open-reading frames. These three functional kappachains were cloned out of the oligoclonal U1-59 hybridoma wellidentified based on V kappa gene usage as (1) VK1 A3-JK2, (2) VK1A20-JK3 and (3) B3-JK1. All V-kappa were cloned into pCDNA expressionvector in frame with a human kappa light chain constant region gene.

Transfections:

Each heavy chain was transfected with each of the kappa chains intransient transfections for a total of 6 heavy chain/kappa light chainpairs. The transfection of the gamma chain with the A20 kappa chain gavepoor antibody expression, while no antibody was secreted or detectedwhen the A20 kappa chain was co-transfected with the mu chain. A totalof three IgG sups and two IgM sups were available for HER-3 bindingassay.

Chain VH D J Constant ORF Heavy VH4-34 D1-20 JH2 Gamma yes Heavy VH2-5D6-6 JH4b Mu yes Light A3 JK2 Kappa yes Light A20 JK3 Kappa yes Light B3JK1 Kappa yes Light A27 JK3 Kappa NO

Binding activity to HER-3+ cell lines was detected in FACS with the IgG1mAb consisting of the VH4-34 and the B3 kappa chain. No other VH/Vkcombinations gave fluorescence signal above background in FACS usingHER-3+ cell lines.

Binding Competition of the Anti-HER-3 Antibodies

Multiplexed competitive antibody binning was performed as published inJia et al. J Immunol Methods. 288, 91-98 (2004) to assess clusters ofHER-3 antibodies that competed for binding to HER-3. Tested HER-3antibodies from cohort 1 clustered into 5 bins based on competition forbinding.

Bin#1 Bin#2 Bin#3 Bin#4 Bin#5 U1-42 U1-48 U1-52 U1-38 U1-45 U1-44 U1-50U1-39 U1-40 U1-62 U1-51 U1-41 U1-46 U1-43 U1-47 U1-49 U1-61 U1-58 U1-53U1-55Epitope Characterization of Anti-HER-3 Antibodies

The epitopes of human anti-HER-3 antibodies of the invention werecharacterized. First a dot blot analysis of the reduced, denaturedHER-3-His tagged purified ECD protein showed absence of binding by theanti-HER-3 antibodies tested (U1-59, U1-61, U1-41, U146, U1-53, U1-43,U1-44, U1-47, U1-52, U1-40, U1-49)) demonstrating that all had epitopessensitive to reduction of disulfide bonds, suggesting that all haddiscontinuous epitopes. Next, the antibodies were mapped to defineddomains in the HER-3 molecule by engineering various human-rat HER-3chimeric molecules, based on the division of the HER-3 extra-cellulardomain into four domains:

1) L1 (D1): the minor ligand-binding domain,

2) S1 (D2): the first cysteine-rich domain,

3) L2 (D3): the major ligand-binding domain, and

4) S2 (D4): the sec cysteine-rich domain.

The extra-cellular domain (ECD) of Human HER-3 cDNA was amplified fromRAT1-HER-3 cells. The rat HER-3 cDNAs was amplified by RT-PCR from ratliver RNA and confirmed by sequencing. The cDNAs expressing the ECD ofhuman and rat Her3 were cloned into mammalian expression vectors asV5-His fusion proteins. Domains from the human HER-3 ECD were swappedinto the scaffold provided by the rat HER-3 ECD by using the Mfe1, BstX1and DraIII internal restriction sites. By this means, various chimericrat/human HER-3 ECD HIS fusion proteins (amino acids 1-160, 161-358,359-575, 1-358, 359-604) were constructed and expressed via transienttransfection of HEK 293T cells. Expression of the constructs wasconfirmed using a rat polyclonal antibody against human HER-3. The humanmonoclonal antibodies were tested in ELISA for binding to the secretedchimeric ECDs.

Two of the human antibodies, including antibody U1-59, cross-reactedwith rat HER-3. To assign binding domains, these mAbs were testedagainst a truncated form of HER-3 consisting of L1-S1-V5his taggedprotein purified from the supernatant of HEK 293T cells transfected witha plasmid DNA encoding the expression of the L1-S1 extra-cellulardomains of HER3. mAb U1-59 bound to the L1-S1 protein in ELISA, implyingthat its epitope is in L1-S1. mAb 2.5.1 did not bind to the L1-S1protein, implying that its epitope is in L2-S2. Further mapping ofantibody U1-59 was accomplished using SELDI time of flight massspectroscopy with on-chip proteolytic digests of mAb-HER-3 ECDcomplexes.

Mapping U1-59 Epitopes Using SELDI

Further mapping of antibody U1-59 was accomplished using a SELDI time offlight mass spectroscopy with on-chip proteolytic digests of mAb-HER-3ECD complexes. Protein A was covalently bound to a PS20 protein chiparray and used to capture mAb U1-59. Then the complex of the PS20protein chip and the monoclonal antibody was incubated with HER-3-Hispurified antigen. Next the antibody-antigen complex was digested withhigh concentration of Asp-N. The chip was washed, resulting in retentionof only the HER-3 peptide bound to the antibody on the chip. The epitopewas determined by SELDI and identified by mass of the fragment. Theidentified 6814 D fragment corresponds to two possible expected peptidesgenerated from a partial digest of the HER-3-his ECD. Both overlappingpeptides map to the domain S1. By coupling SELDI results with binding toa HER-3 deletion construct, the epitope was mapped to residues 251 to325 of SEQ ID NO:390.

The location of the binding domains in the extracellular part of HER-3that are recognized by the human anti-HER-3 mAbs of the invention aresummarized in Table 4. The epitope domain mapping results wereconsistent with results from antibody competition binding competitionbins, with antibodies that cross-competed each other for binding toHER-3 also mapping to the same domains on HER-3 (FIG. 3).

TABLE 4 A summary of mAb's binding domain based on ELISA assay resultsBinding Binding mAb Rat XR domain mAb Rat XR domain U1-59 Yes S1 U1-2 NoL2 U1-61 No L2 U1-7 No L2 U1-41 No L2 U1-9 No L2 U1-46 No S1 U1-10 No L2U1-53 No L2 U1-12 No L2 U1-43 No L2 U1-13 No L2 U1-44 No S1 U1-14 No L2U1-47 No S1 U1-15 No L2 U1-52 Yes L2S2 U1-19 No L2 U1-40 No L2 U1-20 NoL2 U1-49 No L1 U1-21 No L2 U1-21 No L2 U1-28 No L2 U1-22 No L2 (U1-31)No L2 U1-23 No L2 U1-32 No L2 U1-24 No L2 (U1-35) No L2 U1-25 No L2U1-36 No L2 U1-26 No L2 (U1-37) No L2 U1-27 No L2 XR = cross-reactive

Example 9 Determination of Canonical Classes of Antibodies

Chothia, et al. have described antibody structure in terms of “canonicalclasses” for the hypervariable regions of each immunoglobulin chain (J.Mol. Biol., 1987 Aug. 20, 196(4):901-17). The atomic structures of theFab and VL fragments of a variety of immunoglobulins were analyzed todetermine the relationship between their amino acid sequences and thethree-dimensional structures of their antigen binding sites. Chothia, etal. found that there were relatively few residues that, through theirpacking, hydrogen bonding or the ability to assume unusual phi, psi oromega conformations, were primarily responsible for the main-chainconformations of the hypervariable regions. These residues were found tooccur at sites within the hypervariable regions and in the conservedβ-sheet framework. By examining sequences of immunoglobulins havingunknown structure, Chothia, et al. show that many immunoglobulins havehypervariable regions that are similar in size to one of the knownstructures and additionally contained identical residues at the sitesresponsible for the observed conformation.

Their discovery implied that these hypervariable regions haveconformations close to those in the known structures. For five of thehypervariable regions, the repertoire of conformations appeared to belimited to a relatively small number of discrete structural classes.These commonly occurring main-chain conformations of the hypervariableregions were termed “canonical structures.” Further work by Chothia, etal. (Nature, 1989 Dec. 21-28, 342 (6252):877-83) and others (Martin, etal. J. Mol. Biol., 1996 Nov. 15, 263(5): 800-15) confirmed that there isa small repertoire of main-chain conformations for at least five of thesix hypervariable regions of antibodies.

The CDRs of each antibody described above were analyzed to determinetheir canonical class. As is known, canonical classes have only beenassigned for CDR1 and CDR2 of the antibody heavy chain, along with CDR1,CDR2 and CDR3 of the antibody light chain. The tables below summarizesthe results of the analysis. The canonical class data is in the form ofHCDR1-HCDR2-LCDR1-LCDR2-LCDR3, wherein “HCDR” refers to the heavy chainCDR and “LCDR” refers to the light chain CDR. Thus, for example, acanonical class of 1-3-2-1-5 refers to an antibody that has a HCDR1 thatfalls into canonical class 1, a HCDR2 that falls into canonical class 3,a LCDR1 that falls into canonical class 2, a LCDR2 that falls intocanonical class 1, and a LCDR3 that falls into canonical class 5.

Assignments were made to a particular canonical class where there was70% or greater identity of the amino acids in the antibody with theamino acids defined for each canonical class. The amino acids definedfor each antibody can be found, for example, in the articles by Chothia,et al. referred to above. Table 5 and Table 6 report the canonical classdata for each of the HER-3 antibodies. Where there was less than 70%identity, the canonical class assignment is marked with an asterisk(“*”) to indicate that the best estimate of the proper canonical classwas made, based on the length of each CDR and the totality of the data.Where there was no matching canonical class with the same CDR length,the canonical class assignment is marked with a letter s and a number,such as “s18”, meaning the CDR is of size 18. Where there was nosequence data available for one of the heavy or light chains, thecanonical class is marked with “Z”.

TABLE 5 Antibody Antibody (sorted) H1-H2-L1-L2-L3 H3length (sorted)H1-H2-L1-L2-L3 H3length U1-38 3-1-4-1-1 9 U1-7 3-1-2-1-1 12 U1-391-1-4-1*-1 6 U1-9 3-1-2-1-1 12 U1-40 3-1-4-1-1 15 U1-10 3-1-2-1-1 12U1-41 3-1-2-1-1 15 U1-12 3-1-2-1-1 12 U1-42 1-2-2-1-1 9 U1-13 3-1-4-1-17 U1-43 3-1-2-1-1 17 U1-14 3-1-2-1-1 12 U1-44 1-2-2-1-1 9 U1-153-1-8-1-1 14 U1-45 1-2*-2-1-1 16 U1-19 3-1-Z-Z-Z 12 U1-46 3-s18-Z-Z-Z 17U1-20 3-1-2-1-1 19 U1-47 3-s18-2-1-1 16 U1-21 3-1-2-1-1 12 U1-481-1-Z-Z-Z 16 U1-22 3-1-2-1-1 12 U1-49 1-3-4-1-1 17 U1-23 3-1-2-1-1 12U1-50 3-1-2-1-1 17 U1-24 3-1-2-1-1 12 U1-51 1-1-3-1-1 19 U1-25 3-1-2-1-112 U1-52 3-1-8-1-1 15 U1-26 3-1-2-1-1 12 U1-53 1-3-2-1-1 10 U1-273-1-2-1-1 12 U1-55 3-1-4-1-1 15 U1-28 3-1-2-1-1 12 U1-57 3-1-4-1-1 15U1-31 1-2-2-1-1 13 U1-58 1-3-2-1-1 12 U1-32 3-1-2-1-1 12 U1-59 1-1-3-1-19 U1-35 1-3-2-1-1 14 U1-61.1 3-1*-2-1-1 16 U1-36 3-1-2-1-1 12 U1-621-2-8-1-1 12 U1-37 1-2-Z-Z-Z 13 U1-2 3-1-2-1-1 12

Table 7 is an analysis of the number of antibodies per class. The numberof antibodies having the particular canonical class designated in theleft column is shown in the right column. The four mAbs lacking onechain sequence data and thus having “Z” in the canonical assignment arenot included in this counting.

The most commonly seen structure is 3-1-2-1-1: Twenty-one out offorty-one mAbs having both heavy and light chain sequences had thiscombination.

TABLE 6 H1-H2-L1-L2-L3 Count 1-1-3-1-1 2 1-1-4-1*-1 1 1-2-2-1-1 41-2-8-1-1 1 1-3-2-1-1 3 1-3-4-1-1 1 3-1-2-1-1 21 3-1-4-1-1 5 3-1-8-1-1 23-s18-2-1-1 1

Example 10 Determination of Antibody Affinity

Affinity measurements of anti-HER-3 antibodies of the invention wereperformed by indirect FACS Scatchard analysis. Therefore, 10⁵ cells ofinterest or SK-Br 3 cells were harvested with 10 mM EDTA in PBS, washedonce with FACS buffer (PBS, 3% FCS, 0.4% azide) and seeded on a 96-wellround bottom plate. The cells were spun for 3 min at 1000 rpm to removesupernatant and then resuspended with α-HER-3 antibody (3 μg/ml) or withantibody dilutions (100 μl/well) starting with 20 μg/ml human monoclonalantibody in FACS buffer, diluted in 1:2 dilution steps. Cell suspensionswere incubated on ice for 1 hr, washed twice with FACS buffer andresuspended with secondary antibody (100 μl/well) donkey-anti-human-PE(Jackson) diluted 1:50 in FACS buffer. The cell suspensions wereincubated on ice and in the dark for 30 min, washed twice with FACSbuffer and analyzed (FACS, Beckman Coulter). According to the FACSScatchard analysis, the fluorescence mean was calculated for eachmeasurement. Background staining (=without 1^(st) antibody) wassubtracted from each fluorescence mean. Scatchard plot withx-value=fluorescence mean and y-value=fluorescence mean/concentration ofmAb (nM) was generated. The KD was taken as the absolute value of 1/m oflinear equation. FIG. 4 shows a kinetic analysis using the U1-59antibody of the invention. In the following table 8 affinitymeasurements for certain antibodies of the invention selected in thismanner are provided.

TABLE 7 KD clone (nM) U1-38 n.d. U1-39 102 U1-40 6.7 U1-41 0.18 U1-42n.d. U1-43 0.57 U1-44 4 U1-52 16.8 U1-61 0.13 U1-62 20.4 U1-46 13.8U1-47 9.38 U1-49 1 U1-50 39.3 U1-51 131.6 U1-53 0.082 U1-55.1 3.7 U1-586.4 U1-59 3.69 U1-24 0.06 U1-7 0.02

Example 11 Anti-HER-3 Antibodies of the Invention Induce HER-3 ReceptorEndocytosis

HER-3 has been identified as a factor that can influence initiation andprogression of hyperproliferative diseases through serving as animportant gatekeeper of HER family mediated cell signaling. Thus, ifHER-3 is effectively cleared from the cell surface/membrane by receptorinternalization, cell signaling and therefore transformation and/ormaintenance of cells in malignancy can be ultimately diminished orsuppressed.

In order to investigate whether anti-HER-3 antibodies of the inventionare capable of inducing accelerated endocytosis of HER-3, the relativeamount of HER-3 molecules on the cell surface after 0.5 and 4 hrincubation of the cells with anti-HER-3 antibodies of the invention werecompared. 3×10⁶ cells were seeded in normal growth medium in 24-welldish and left to grow overnight. Cells were preincubated with 10 μg/mlanti-HER-3 mAbs in normal growth medium for the indicated times at 37°C. Cells were detached with 10 mM EDTA and incubated with 10 μg/mlanti-HER-3 mAbs in wash buffer (PBS, 3% FCS, 0.04% azide) for 45 min at4° C. Cells were washed twice with wash buffer, incubated withdonkey-anti-human-PE secondary antibody (Jackson) diluted 1:100 for 45min at 4° C., washed twice with wash buffer and analyzed by FACS(BeckmanCoulter, EXPO).

Data shown in FIG. 5 demonstrate that treatment of cells with anti-HER-3antibodies leads to internalization of the receptor. Data are shown as %internalization and refer to the reduction of the mean fluorescenceintensity of anti-HER3 treated samples relative to control-treatedsamples.

Example 12 Inhibition of Ligand Binding to Human Cancer Cells SKBr3 byHuman Anti-HER-3 Antibodies of the Invention

Radioligand competition experiments were performed in order toquantitate the ability of the anti-HER-3 antibodies of the invention toinhibit ligand binding to HER-3 in a cell based assay. Therefore, theHER-3 receptor binding assay was performed with 4×10⁵ SK-BR-3 cellswhich were incubated with varying concentrations of antibodies for 30min on ice. 1.25 nM [I¹²⁵]-α-HRG/[¹²⁵I]-β-HRG were added to each welland the incubation was continued for 2 hr on ice. The plates were washedfive times, air-dried and counted in a scintillation counter. FIGS. 6a-e show the results of these experiments performed with representativeanti-HER-3 antibodies of the invention and demonstrate that theantibodies of the invention are capable of specifically reducing thebinding of [¹²⁵I]-α-HRG/[¹²⁵I]-β-HRG to cells expressing endogenousHER-3.

Example 13 Inhibition of Ligand-Induced HER-3 Phosphorylation by HumanAnti-HER-3 Antibodies of the Invention

ELISA experiments were performed in order to investigate whether theantibodies of the invention are able to block ligand β-HRG-mediatedactivation of HER-3. Ligand-mediated HER-3 activation was detected byincreased receptor tyrosine phosphorylation.

Day 1: 1×96 well dish was coated with 20 μg/ml Collagen I in 0.1 Macetic acid for 4 hr at 37° C. 2.5×10⁵ cells were seeded in normalgrowth medium

Day 2: Cells were starved in 100 μl serum free medium for 24 hr.

Day 3: Cells were preincubated with 10 μg/ml anti-HER-3 mAbs for 1 hr at37° C. and then treated with 30 ng/ml β-HRG-EGF domain (R&D Systems) for10 min. Medium was flicked out and cells were fixed with 4% formaldehydesolution in PBS for 1 hr at room temperature. Formaldehyde solution wasremoved and cells were washed with wash buffer (PBS/0.1% Tween 20).Cells were quenched with 1% H₂O₂, 0.1% NaN₃ in wash buffer and incubatedfor 20 min at room temperature, then blocked with NET-Gelantine for 5 hrat 4° C. Primary antibody phospho-HER-3 (Tyr1289) (polyclonal rabbit;Cell signaling #4791; 1:300) was added overnight at 4° C.

Day 4: The plate was washed 3× with wash buffer, then incubated withanti-rabbit-POD diluted 1:3000 in PBS-0.5% BSA was added to each welland incubated for 1.5 hr at room temperature. The plate was washed 3×with wash buffer and once with PBS. Tetramethylbenzidine (TMB,Calbiochem) was added and monitored at 650 nm. The reaction was stoppedby addition of 100 μl 250 nM HCl and the absorbance was read at 450 nmwith a reference wavelength of 650 nm using a Vmax plate reader (ThermoLab Systems).

FIG. 7 a shows representative results of this experiment, demonstratingthat anti-HER-3 antibodies of the invention were able to reduceligand-mediated HER-3 activation as indicated by decreased receptortyrosine phosphorylation. Data are shown as percent reduction bytherapeutic antibodies relative to a control antibody.

To test potency of mAb U1-53 to inhibit ligand induced HER-3 activation,MCF-7 cells were starved for 24 hr, incubated with mAb U1-53 for 1 hr at37° C. and stimulated with 10 nM HRG-β for 10 min. Lysates weretransferred to 1B4 (mouse anti-HER-3 mAb) ELISA plates andphosphorylation of HER-3 was analysed with antibody 4G10. As shown inFIG. 1 b phosphorylation of HER-3 was almost completely inhibited in adose dependent manner with an IC50 of 0.14 nM.

Example 14 Inhibition of Ligand-Induced p42/p44 MAP-KinasePhosphorylation by Human Anti-HER-3 Antibodies of the Invention

Next ELISA experiments were performed in order to investigate whetherthe antibodies of the invention are able to block ligand β-HRG-mediatedactivation of p42/p44 MAP-Kinase. Ligand-mediated HER-3 activation wasdetected by increased protein (Thr202/Tyr204) phosphorylation.

Day 1: 1×96 well dish was coated with 20 μg/ml Collagen I in 0.1 Macetic acid for 4 hr at 37° C. 3×10⁵ cells were seeded in normal growthmedium

Day 2: Cells were starved in 100 μl serum free medium for 24 hr.

Day 3: Cells were preincubated with 5 μg/ml anti-HER-3 mAbs for 1 hr at37° C. and then treated with 20 ng/ml β-HRG-EGF domain (R&D Systems) for10 min. Medium was flicked out and cells were fixed with 4% formaldehydesolution in PBS for 1 hr at room temperature. Formaldehyde solution wasremoved and cells were washed with wash buffer (PBS/0.1% Tween 20).Cells were quenched with 1% H₂O₂, 0.1% NaN₃ in wash buffer and incubatedfor 20 min at room temperature, then blocked with PBS/0.5% BSA for 5 hrat 4° C. Primary antibody phospho-p44/p42 MAP Kinase (Thr202/Tyr204)(polyclonal rabbit; Cell signaling #9101; 1:3000) was added overnight at4° C.

Day 4: The plate was washed 3× with wash buffer, then incubated withanti-rabbit-HRP diluted 1:5000 in PBS-0.5% BSA was added to each welland incubated for 1.5 hr at room temperature. The plate was washed 3×with wash buffer and once with PBS. Tetramethylbenzidine (TMB,Calbiochem) was added and monitored at 650 nm. The reaction was stoppedby addition of 100 μl 250 nM HCl and The absorbance was read at 450 nmwith a reference wavelength of 650 nm using a Vmax plate reader (ThermoLab Systems).

FIG. 8 shows representative results of this experiment. The antibodiesof the invention were able to reduce ligand-mediated p42/p44 MAP-Kinaseactivation as indicated by decreased phosphorylation. Data are shown aspercent reduction by therapeutic antibodies relative to a controlantibody.

Example 15 Inhibition of β-HRG-Induced Phospho-AKT Phosphorylation byHuman Anti-HER-3 Antibodies of the Invention

In the following ELISA experiment we investigated whether the anti-HER-3antibodies of the invention are able to block ligand β-HRG-mediatedactivation of AKT-Kinase. Ligand-mediated AKT activation was detected byincreased protein (Ser473) phosphorylation.

Day 1: 1×96 well dish was coated with 20 μg/ml Collagen I in 0.1 Macetic acid for 4 hr at 37° C. 3×10⁵ cells were seeded in normal growthmedium

Day 2: Cells were starved in 100 μl serum free medium for 24 hr.

Day 3: Cells were preincubated with 5 μg/ml anti-HER-3 mAbs for 1 hr at37° C. and then treated with 20 ng/ml β-HRG-EGF domain (R&D Systems) for10 min. Medium was flicked out and cells were fixed with 4% formaldehydesolution in PBS for 1 hr at room temperature. Formaldehyde solution wasremoved and cells were washed with wash buffer (PBS/0.1% Tween 20).Cells were quenched with 1% H₂O₂, 0.1% NaN₃ in wash buffer and incubatedfor 20 min at room temperature, then blocked with PBS/0.5% BSA for 5 hrat 4° C. Primary antibody phospho-Akt (Ser473) (polyclonal rabbit; Cellsignaling #9217; 1:1000) was added overnight at 4° C.

Day 4: The plate was washed 3× with wash buffer, then incubated withanti-rabbit-HRP diluted 1:5000 in PBS-0.5% BSA was added to each welland incubated for 1.5 hr at room temperature. The plate was washed 3×with wash buffer and once with PBS. Tetramethylbenzidine (TMB,Calbiochem) was added and monitored at 650 nm. The reaction was stoppedby addition of 100 μl 250 nM HCl and The absorbance was read at 450 nmwith a reference wavelength of 650 nm using a Vmax plate reader (ThermoLab Systems).

FIG. 9 shows representative results of this experiment. The anti-HER-3antibodies of the invention were able to reduce β-HRG-mediated AKT asindicated by decreased phosphorylation. Data are shown as percentreduction by therapeutic antibodies relative to a control antibody.

Example 16 Inhibition of α-HRG/β-HRG-Mediated MCF7 Cell Proliferation byHuman Anti-HER-3 Antibodies of the Invention

In vitro experiments were conducted in order to determine the ability ofthe antibodies of the invention to inhibit HRG-stimulated cellproliferation. 2000 MCF7 cells were seeded in FCS-containing medium on96-well plates overnight. Cells were preincubated in quadruplicates withantibody diluted in medium with 0.5% FCS for 1 hr at 37° C. Cells werestimulated with 30 ng/ml α- or 20 ng/ml β-HRG (R&D Systems) by addingligand directly to antibody solution and were then left to grow for 72hr. AlamarBlue™ (BIOSOURCE) was added and incubated at 37° C. in thedark. Absorbance was measured at 590 nm every 30 min. The data weretaken 90 min after addition of alamar blue. The results as indicated inFIG. 10 show that representative antibodies of the invention inhibitHRG-induced cell growth in human cancer cells. Data are shown as percentreduction by therapeutic antibodies relative to a control antibody.

Example 17 Inhibition of β-HRG-Induced MCF7 Cell Migration by HumanAnti-HER-3 Antibodies of the Invention

Transmigration experiments were performed in order to investigatewhether the antibodies of the invention block cell migration.Serum-starved MCF7 cells were preincubated by adding the indicatedamount of antibody to the cell suspension and incubating both for 45 minat 37° C. 500 μl cell suspension (50,000 cells) was then placed in thetop chamber of collagen 1-coated transwells (BD Falcon, 8 μm pores). 750μl medium (MEM, amino acids, Na-pyruvate, Pen.-Strept., 0.1% BSA,without fetal calf serum) alone or containing the ligands β-HRG-EGFdomain (R&D Systems) were used in the bottom chamber. Cells were left tomigrate for 8 hr at 37° C. and were stained with DAPI.

Stained nuclei were counted manually; percent inhibition was expressedas inhibition relative to a control antibody.

FIG. 11 shows the result of the experiment demonstrating thatrepresentative anti-HER-3 antibodies of the invention reduce HRG-inducedcell migration.

Example 18 Colony Formation Assay (Soft Agar Assay)

Soft agar assays were conducted in order to investigate the ability ofthe anti-HER-3 antibodies of the invention to inhibit anchorageindependent cell growth. The soft agar colony formation assay is astandard in vitro assay to test for transformed cells, as only suchtransformed cells can grow in soft agar.

750 to 2000 cells (depending on the cell line) were preincubated withindicated antibodies at 10 μg/ml in IMDM medium (Gibco) for 30 min andresuspended in 0.4% Difco noble agar. The cell suspension was plated on0.75% agarose underlayer containing 20% FCS in quadruplicate in a96-well plate. Colonies were allowed to form for 14 days and were thenstained with 50 μl MTT (0.5 mg/ml in PBS) overnight. FIGS. 12 a-i showthe results of these experiments performed with three representativeantibodies of the invention. These results demonstrate that anti-HER-3antibodies of the invention reduce anchorage independent cell growth ofMDA-MB361 and NCI-ADR breast cancer cells (FIG. 12 a,b), MKN-28 gastriccancer (FIG. 12 c), HT144 melanoma cells (FIG. 12 d), Skov3 ovarycarcinoma cells (FIG. 12 e), PPC-1 prostate cancer cells (FIG. 12 f),BX-PC3 pancreas cancer cells (FIG. 12 g), A431 epidermoid carcinomacells (FIG. 12 h) and lung carcinoma cells (FIG. 12 i). Colonies werecounted with a Scanalyzer HTS camera system (Lemnatec, Wuerselen).

Example 19 Human Anti-HER-3 Antibodies Inhibit Human Breast CarcinomaGrowth in Nude Mice

The anti-tumor efficacy of therapeutic antibodies is often evaluated inhuman xenograft tumor studies. In these studies, human tumors grow asxenografts in immunocompromised mice and therapeutic efficacy ismeasured by the degree of tumor growth inhibition. In order todetermine, if the anti-HER-3 antibodies of the invention interfere withtumor growth of human breast cancer cells in nude mice, 5×10⁶ T47D cellswere implanted in female NMRI nude/nude mice. Tumors were subcutaneous,grown on the back of the animal. Treatments began when tumors reached amean volume of 20 mm³; eight days post implantation. Prior to firsttreatment, mice were randomized and statistical tests performed toassure uniformity in starting tumor volumes (mean, median and standarddeviation) across treatment groups. Treatment started with a loadingdose of 50 mg/kg followed by 25 mg/kg injections once a week byintraperitoneal injection. A control arm received doxorubicin(pharmaceutical grade). All animals were supplemented with 0.5mg/kg/week oestrogen injected i.p.

Details of the treatment groups are given below.

Loading Weekly dose Gr. N 1^(st) Compound (mg/kg) (mg/kg) RouteSchedule 1. 10 PBS — i.p. once/week 2. 10 doxorubicin  8 mg/kg i.v.once/week* 3. 10 U1-53 50 mg/kg 25 mg/kg i.p. once/week 20 ml/kg 10ml/kg *doxorubin treatment as described by Boven et al., CancerResearch, 1992.

Data for median tumor volume (FIG. 13) demonstrated that administrationof an anti-HER-3 antibody of the invention resulted in reduction oftumor growth.

Example 20 Human Anti-HER-3 Antibodies Inhibit Human Pancreatic TumorGrowth in SCID Mice

To test the therapeutic potential of anti-HER3 antibodies in other solidtumor types the anti-HER-3 antibodies, U1-53 and U1-59, were tested inmice with established tumors derived from the human pancreatic tumorcell line BxPC3. As controls sets of mice treated with either thevehicle control, PBS, or the established therapeutic antibody, Erbitux,were included. 5×10⁶ BxPC3 cells were inoculated subcutaneously withoutMatrigel into CB17 SCiD mice. Mice bearing established tumors with amean volume of 140 mm² received 50 mg/kg of U1-53, U1-59, Erbitux or theequivalent volume of PBS via intraperitoneal injection. Thereafter themice received once weekly 25 mg/kg injections for the duration of thestudy.

The results for this experiment are shown in FIG. 14. U1-53 and U1-59reduced the growth of the human pancreatic tumors in a cytostaticfashion. Notably, in this experiment, U1-53 and U1-59 were moreeffective than the EGFR-targeting antibody Erbitux at delaying tumorgrowth. These data demonstrated the therapeutic efficacy of anti-HER-3antibodies of the invention in comparison to a benchmark therapeuticagent.

Example 21 Combining the Human Anti-HER-3 Antibodies with Anti-EGFRAntibodies Increases Anti-Tumor Activity

The monotherapy of hyperproliferative diseases with targeted antibodiesis often hampered by problems such as, on the one hand, the developmentof resistance to drugs, and on the other hand, a change in theantigenicity. For example, loss of antigenicity after prolongedtreatment may render tumor cells insensitive to therapeutic antibodies,since those tumor cells that do not express or have lost the targetedantigen have a selective growth advantage. These problems might beevaded by using the antibodies of the invention in combination with atherapeutic antibody that targets a different receptor on the tumorcells, or another antineoplastic agent. Intervening in multiplesignaling pathways or even related pathways but at multiple interventionsteps might also provide therapeutic benefit. These combined treatmentmodalities are likely to be more efficacious, because they combine twoanti-cancer agents, each operating via a different mechanism of action.

In order to demonstrate the feasibility of the anti-HER-3 antibodies ofthe invention, U1-53 and U1-59, as suitable combination agents, wecompared monotherapeutic administrations of U1-53 or U1-59 with those inwhich either U1-53 or U1-59 was combined with the anti-EGR specificantibody, Erbitux. 5×10⁶ BxPC3 cells were inoculated subcutaneously withMatrigel into CB17 SCID mice. After tumor volumes had reached 200 mm³,mice were randomized into individual treatment groups. Weeklyintraperitoneal administrations of U1-53, U1-59 and Erbitux as singleagents or combinations of either anti-HER3 antibodies with Erbitux or asa cocktail of two anti HER-3 antibodies were performed. All antibodieswere dosed at a single loading doses of 50 mg/kg/week, followed byweekly injections of 25 mg/kg for six weeks. Control arms receivedbi-weekly administrations of Gemcitabine (120 mg/kg), weekly pooledhuman IgG or weekly vehicle (PBS) injections. The regimens are detailedbelow.

Loading Weekly dose dose Gr. N Compound (mg/kg) (mg/kg) Route Schedule4. 12 PBS 20 ml/kg 10 ml/kg q7d i.p. 5. 12 Pooled human 50 mg/kg 25mg/kg q7d i.p. IgG 6. 12 U1-53 50 mg/kg 25 mg/kg q7d i.p. 7. 12 U1-59 50mg/kg 25 mg/kg q7d i.p. 8. 12 Erbitux 50 mg/kg 25 mg/kg q7d i.p. 9. 12U1-53 + 25 mg/kg 12.5 mg/kg   q7d i.p. Erbitux each each 10. 12 U1-59 +25 mg/kg 12.5 mg/kg   q7d i.p. Erbitux each each 11. 12 U1-53 + U1-59 25mg/kg 12.5 mg/kg   q7d i.p. each each 12. 12 Gemcitabine none 120 mg/kg 2x ip weekly

The results for this experiment are shown in FIG. 15. Antibodies U1-53and U1-59, when administered as single agents, delayed the growth of thehuman pancreatic tumors to the same degree as Gemcitabine, which isoften used as a standard anti-pancreatic cancer chemotherapy.Co-administration of Erbitux with U1-53 or U1-59 resulted in asignificantly greater reduction of tumor growth than observed witheither single agent administration of U1-53, U1-59 or Erbitux. Thus, abeneficial therapeutic response can be achieved by combining theanti-HER-3 antibodies of the invention with suitable antibodies thattarget separate tumor antigens.

In summary, the anti-HER-3 antibodies of the invention have potenttherapeutic efficacy against human tumors in vivo. They can beeffectively combined with other anti-neoplastic therapeutics forincreased anti-tumor activity.

Example 22 Human Anti-HER-3 Antibodies Inhibit Human Melanoma TumorGrowth in nu/nu Mice

Members of the erbB family of receptors, including Her3, are abnormallyexpressed in a large variety of epithelial cancers and they are known toplay important roles in the growth and survival of many these solidtumors. These tumors include melanomas, head and neck squamous cellcancers, non-small cell lung cancers and prostate, glioma, gastric,breast, colorectal, pancreatic, ovarian cancers. In order to verify,that the anti-Her3 antibodies of the invention are not restricted intheir anti-cancer activity to individual tumor types, e.g. pancreaticcancers (see Example 21), but can be used as therapeutics against manyHER-3-dependent tumors, we tested U1-53 and U1-59 in additionalxenograft studies. One example is shown in FIG. 16. 5×10⁵ human melanomacells, HT144, were injected subcutaneously into CB17 SCID mice, followedby immediate subsequent intraperitoneal injection of 50 mg/kg of U1-53and U1-59, the equivalent volume of PBS or Dacarbacin (DITC) at 200mg/kg. Thereafter, mice received 25 mg/kg of U1-53 or U1-59 once weekly,whereas DITC was given once every two weeks at 200 mg/kg.

The median tumor volumes from each treatment group are shown in FIG. 16.Administration of the antibodies of the invention resulted in growthreduction of the human melanomas when compared to tumors that had beentreated with the vehicle control. These results demonstrate that theantibodies of the invention are not restricted in their therapeuticpotential and target a wide variety of HER-3 expressing cancers.

Example 23 Human Anti-HER-3 Antibodies Inhibit Growth of Colon CarcinomaXenografts in Mice

HT-29 human colon carcinoma cells were suspended in medium with 2:1ratio of Matrigel to a final concentration of 10×10⁶ cells/ml. 0.2 ml ofcell suspension were injected s.c. into the right flank of 4-5-week-oldCD1 nu/nu mice. A total of 95 mice were used.

The mice were randomly assigned to control and treatment groups. Thetreatment started on the same day. Duration of treatment was 29 days.Upon completion of the study, three tumours per group were collected 3hours after administration of treatment. The tumours were fast-frozenand kept at −80° C.

The following treatment protocol was carried out:

Control group: non-specific human IgG 25 mg/kg, twice weekly,intraperitoneal

Treatment group: antibody U1-53, 25 mg/kg, twice weekly, intraperitoneal

Treatment group: antibody U1-7, 25 mg/kg, twice weekly, intraperitoneal

Treatment group: antibody U1-59, 25 mg/kg, twice weekly, intraperitoneal

Treatment group 5-FU: 5-fluorouracil, 50 mg/kg, 9 d×5, intraperitoneal

The median tumor volumes from each group are shown in FIG. 17.Administration of the antibodies of the invention resulted in growthreduction of the HT-29 colon carcinoma tumors when compared to tumorsthat had been treated with non-specific human IgG1.

Example 24 Human Anti-HER-3 Antibodies Inhibit Lung Cancer Growth inMice

Calu-3 human non-small cell lung cancer cells were suspended in mediumwith 1:1 ratio of Matrigel to a final concentration of 5×10⁶ cells/ml.0.05 ml of cell suspension were injected s.c. into the right flank of9-week-old female CB17 scid mice. A total of 60 mice were used.

The mice were randomly selected to control and treatment groups.Treatment started on the same day. The duration of treatment was 32days.

The following treatment protocol was carried out:

PBS Vehicle Group

hG control group: non-specific human IgG: 25 mg/kg, twice weekly,intraperitoneal

Treatment group antibody U1-53, 25 mg/kg, twice weekly, intraperitoneal

Treatment group antibody U1-7, 25 mg/kg, twice weekly, intraperitoneal

Treatment group antibody U1-59, 25 mg/kg, twice weekly, intraperitoneal

The median tumor volumes from each control and treatment group are shownin FIG. 18. Administration of the antibodies of the invention resultedin growth reduction of the human non-small lung cancer xenografts whencompared to tumors that had been treated with the PBS vehicle control ornon-specific human IgG.

Example 25 Human Anti-HER-3 Antibodies Inhibit Human Pancreatic TumorGrowth in Balb/C-Mice

Human pancreatic BxPC3 tumor cells were suspended in medium with a 2:1ratio of Matrigel to a final concentration of 5×10⁶ cells per ml. 0.2 mlof cell suspension were injected s.c. into the right flank of5-7-week-old female Balb/c nu/nu mice. A total of 100 mice were used.

The mice were randomly distributed into control and treatment groups.The treatment started on the same day. The treatment duration was 27days.

The following treatment protocol was carried out:

hIgG control group: non-specific human IgG2, 25 mg/kg, twice weekly,intraperitoneal

Treatment group antibody U1-53, 25 mg/kg, twice weekly, intraperitoneal

Treatment group antibody U1-7, 25 mg/kg, twice weekly, intraperitoneal

Treatment group antibody U1-59, 25 mg/kg, weekly, intraperitoneal

Gemzar treatment group, gemcitabine, 80 mg/kg, weekly, intraperitoneal

The median tumor volumes from each control and treatment group are shownin FIG. 19. Administration of the antibodies of the invention resultedin growth reduction of the human pancreatic tumors when compared totumors that had been treated with non-specific human IgG or with Gemzar.

The inhibition of HER-3 in the human pancreatic tumors could also beshown in a pharmacodynamic experiment. The BxPC3 tumor xenografts weregrown as described above. 3 mice were treated with 500 μg of an IgG1control antibody and 3 mice were treated with 500 μg of the anti-HER-3antibody U1-59. The mice were treated on day 1 and day 4 and thensacrificed on day 5 to measure the antibody-dependent inhibition ofHER-3 phosphorylation (pHER-3).

The tumors were homogenized in a standard RIPA buffer with proteaseinhibitors. 50 μg clear lysate was separated on a 4-20% Tris-glycinegel, transferred onto a nitrocellulose membrane and blocked in 3% bovineserum albumin (BSA). Immunoblotting was performed using an anti-pHER-3antibody (antibody 21D3, Cell Signaling technology). An anti-actinantibody (AB a-2066, Sigma) was used as a control.

The expression was detected by enhanced chemiluminescence (AmershamBiosciences, Piscataway, N.J.). The images were captured with theVersadoc 5000 Imaging System (BioRad, Hercules, Calif.).

The results are shown in FIG. 20. After administration of the humananti-HER-3-antibody U1-59 phosphorylation of HER-3 was no longerdetectable. Thus, the antibodies of the invention are capable ofsignificantly reducing HER-3 activation in human pancreatic tumor cells.

Example 26 Use of Anti-HER-3 Antibodies of the Invention as a DiagnosticAgent

Anti-HER-3 mAb can be used in the diagnostic of malignant diseases.HER-3 is expressed on tumor cells in a very distinct way compared tonormal tissue and, therefore, an expression analysis of HER-3 wouldassist in the primary diagnosis of solid tumors, staging and grading ofsolid tumors, assessment of prognostic criteria for proliferativediseases and neoplasias and risk management in patients with HER-3positive tumors.

A. Detection of HER-3 Antigen in a Sample

An Enzyme-Linked Immunosorbent Assay (ELISA) for the detection of HER-3antigen in a sample is developed. In the assay, wells of a microtiterplate, such as a 96-well microtiter plate or a 384-well microtiterplate, are adsorbed for several hr with a first fully human monoclonalantibody directed against the HER-3 antigen. The immobilized antibodyserves as a capture antibody for any of the HER-3 antigen that may bepresent in a test sample. The wells are rinsed and treated with ablocking agent such as milk protein or albumin to prevent nonspecificadsorption of the analyte.

Subsequently the wells are treated with a test sample suspected ofcontaining the HER-3 antigen, or with a solution containing a standardamount of the HER-3 antigen. Such a sample is, for example, a serumsample from a subject suspected of having levels of circulating HER-3antigen considered to be diagnostic of a pathology. After rinsing awaythe test sample or standard, the wells are treated with a second fullyhuman monoclonal anti-HER-3 antibody of the invention that is labelledby conjugation with biotin. The labeled anti-HER-3 antibody serves as adetecting antibody. After rinsing away excess secondary antibody, thewells are treated with avidin-conjugated horseradish peroxidase (HRP)and a suitable chromogenic substrate. The concentration of the HER-3antigen in the test samples is determined by comparison with a standardcurve developed from the standard samples.

B. Detection of HER3-Antigen in Immunohistochemistry (IHC)

In order to determine HER3-antigen in tissue sections by IHC,Paraffin-embedded tissues are first deparaffinized in xylene for 2×5 minand then hydrated with 100% Ethanol 2×3 min, 95% Ethanol 1 min andrinsed in distilled water. Antigenic epitopes masked byformalin-fixation and paraffin-embedding are exposed by epitopeunmasking, enzymatic digestion or saponin. For epitope unmaskingparaffin sections are heated in a steamer, water bath or microwave ovenfor 2040 min in a epitope retrieval solution as for example 2N HClsolution (pH 1.0). In the case of an enzyme digestion, tissue sectionsare incubated at 37° C. for 10-30 minutes in different enzyme solutionssuch as proteinase K, trypsin, pronase, pepsin etc.

After rinsing away the epitope retrieval solution or excess enzyme,tissue sections are treated with a blocking buffer to prevent unspecificinteractions. The primary antibody is incubated at appropriate dilutionsin dilution buffer for 1 hour at room temperature or overnight. Excessprimary antibody is rinsed away and sections are incubated in peroxidaseblocking solution for 10 min at room temperature. After another washingstep, tissue sections are incubated with a secondary antibody labelledwith a group that might serve as an anchor for an enzyme. Examplestherefore are biotin labelled secondary antibodies that are recognizedby streptavidin coupled horseradish peroxidase. Detection of saidantibody/enzyme complex is achieved by incubating with a suitablechromogenic substrate.

C. Determination of HER-3 Antigen Concentration in Serum of Patients

A sandwich ELISA is developed to quantify HER-3 levels in human serum.The two fully human monoclonal anti-HER-3 antibodies used in thesandwich ELISA, recognized different domains on the HER-3 molecule anddo not compete for binding, for example, see Example 8. The ELISA isperformed as follows: 50 μl of capture ant-HER-3 antibody in coatingbuffer (0.1 M NaHCO₃, pH 9.6) at a concentration of 2 μg/ml were coatedon ELISA plates (Fisher). After incubation at 4° C. overnight, theplates are treated with 200 μl of blocking buffer (0.5% BSA, 0.1% Tween20, 0.01% Thimerosal in PBS) for 1 hr at 25° C. The plates were washed(3×) using 0.05% Tween 20 in PBS (washing buffer, WB). Normal or patientsera (Clinomics, Bioreclaimation) are diluted in blocking buffercontaining 50% human serum. The plates are incubated with serum samplesovernight at 4° C., washed with WB, and then incubated with 100 μl/wellof biotinylated detection anti-HER-3 antibody for 1 hr at 25° C. Afterwashing, the plates are incubated with HRP-Streptavidin for 15 min,washed as before, and then treated with 100 μl/well ofo-phenylenediamine in H₂O₂ (Sigma developing solution) for colorgeneration. The reaction is stopped with 50 μl/well of H₂SO₄ (2 M) andanalyzed using an ELISA plate reader at 492 nm. The concentration ofHER-3 antigen in serum samples is calculated by comparison to dilutionsof purified HER-3 antigen using a four parameter curve fitting program.

Staging of Cancer in a Patient

Based on the results set forth and discussed under items A, B and C,through use of the present invention, it is possible to stage a cancerin a subject based on expression levels of the HER-3 antigen. For agiven type of cancer, samples of blood are taken from subjects diagnosedas being at various stages in the progression of the disease, and/or atvarious points in the therapeutic treatment of the cancer. Theconcentration of the HER-3 antigen present in the blood samples isdetermined using a method that specifically determines the amount of theantigen that is present. Such a method includes an ELISA method, such asthe method described under items A. and B. Using a population of samplesthat provides statistically significant results for each stage ofprogression or therapy, a range of concentrations of the HER-3 antigenthat may be considered characteristic of each stage is designated.

In order to stage the progression of the cancer in a subject understudy, or to characterize the response of the subject to a course oftherapy, a sample of blood is taken from the subject and theconcentration of the HER-3 antigen present in the sample is determined.The concentration so obtained is used to identify in which range ofconcentrations the value falls. The range so identified correlates witha stage of progression or a stage of therapy identified in the variouspopulations of diagnosed subjects, thereby providing a stage in thesubject under study.

Example 27 Uses of Anti-HER-3 Antibodies and Antibody Conjugates of theInvention for Treatment or Prevention of Hyperproliferative Diseases

Many solid tumors are driven by HER family mediated signalling and ithas been demonstrated that HER-3 is a crucial partner through complexformation between HER-1, HER-2 and HER-4. Therefore, a reduction orelimination of HER-3 mediated signaling would impact all other HERfamily members and impair cell signaling leading to a wide window oftherapeutic interventions and potential in combination therapy withother targeted agents, biologics and cytotoxic agents. Thus, anti-HER-3antibodies of the invention can be used for treatment of certainhyperproliferative or HER-3 associated disorders, that are based on anumber of factors as for example HER-3 expression. Tumor types as breastcancer, gastrointestinal cancer, pancreas cancer, prostate cancer,ovarian cancer, stomach cancer, endometrial cancer, salivary glandcancer, lung cancer, kidney cancer, colon cancer, colorectal cancer,thyroid cancer, bladder cancer, glioma, melanoma, other HER-3 expressingor overexpressing cancers, appear to present preferred indications, butindications are not limited to those on the preceding list. In additionthe following groups of patients will benefit from anti-HER-3 directedmAb treatment:

-   -   Patients with resistance to anti-HER-2 mAb treatment    -   Patients not eligible for the treatment with anti-HER-2 mAb    -   Patients with resistance to anti-HER-1 mAb or small molecule        anti-EGFR inhibitor    -   Patients with non-small cell lung cancer resistant to erlotinib        or gefitinib.

Anti-HER-3 antibodies of the invention would be used either as amonotherapy or in combination with one or more agent in a so called“combination therapy”. Said combination therapy may include, but is notlimited to, agents that were specified previously in the invention.Combination therapy with anti-HER3 antibodies and other agents mayextend patient survival, time to tumor progression or quality of patientlife. Protocol and administration design will address therapeuticefficacy as well as the ability to reduce the usual doses of standardtherapies, as for example chemo- or radiation therapy.

Treatment of Humans with Anti-HER-3 Antibodies of the Invention

To determine the in vivo effects of anti-HER-3 antibody treatment inhuman patients with tumors, such human patients are injected over acertain amount of time with an effective amount of anti-HER-3 antibodyof the invention. At periodic times during the treatment, the humanpatients are monitored to determine whether their tumors progress, inparticular, whether the tumors grow and metastasize.

A tumor patient treated with the anti-HER-3 antibodies of the inventionhas a lower level of tumor growth and/or metastasis compared to thelevel of tumor growth and metastasis in tumor patients treated with thecurrent standard of care therapeutics.

Treatment with Anti-HER-3 Antibody Conjugates of the Invention

To determine the in vivo effects of anti-HER-3 antibody conjugates ofthe invention, human patients or animals exhibiting tumors are injectedover a certain amount of time with an effective amount of anti-HER-3antibody conjugate of the invention. For example, the anti-HER-3antibody conjugate administered is DM1-anti-HER-3 antibody conjugate, anauristatin-anti-HER-3 antibody conjugate or radioisotope-anti-HER-3antibody conjugate. At periodic times during the treatment, the humanpatients or animals are monitored to determine whether their tumorsprogress, in particular, whether the tumors grow and metastasize.

A human patient or animal exhibiting tumors and undergoing treatmentwith, for example, DM1-anti-HER-3 antibody or radioisotope-anti-HER-3antibody conjugates has a lower level of tumor growth and metastasiswhen compared to a control patient or animal exhibiting tumors andundergoing treatment with an alternate therapy. Control DM1-antibodiesthat may be used in animals include conjugates comprising DM1 linked toantibodies of the same isotype of the anti-HER-3 antibodies of theinvention, but more specifically, not having the ability to bind toHER-3 tumor antigen. Control radioisotope-antibodies that may be used inanimal tests include conjugates comprising radioisotope linked toantibodies of the same isotype of the anti-HER-3 antibodies of theinvention, but more specifically, not having the ability to bind toHER-3 tumor antigen. Note: the control conjugates would not beadministered to humans.

General Remarks

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the construct deposited,since the deposited embodiment is intended as a single illustration ofcertain objects of the invention and any constructs that arefunctionally equivalent are within the scope of this invention. Thedeposit of material herein does not constitute an admission that thewritten description herein contained is inadequate to enable thepractice of any object of the invention, including the best modethereof, nor is it to be construed as limiting the scope of the claimsto the specific illustrations that it represents.

The foregoing description and Examples detail certain preferredembodiments of the invention and describes the best mode contemplated bythe inventors. It will be appreciated, however, that no matter howdetailed the foregoing may appear in text, the invention may bepracticed in many ways and the invention should be construed inaccordance with the appended claims and any equivalents thereof.

Furthermore, unless otherwise defined, scientific and technical termsused in connection with the present invention shall have the meaningsthat are commonly understood by those of ordinary skill in the art.Moreover, unless otherwise required by context, singular terms shallinclude pluralities and plural terms shall include the singular.Generally, nomenclatures utilized in connection with, and techniques of,cell and tissue culture, molecular biology, and protein and oligo- orpolynucleotide chemistry and hybridization described herein are thosewell known and commonly used in the art. Standard techniques are usedfor recombinant DNA, oligonucleotide synthesis, and tissue culture andtransformation (e.g. electroporation, lipofection). Enzymatic reactionsand purification techniques are performed according to manufacturer'sspecifications or as commonly accomplished in the art or as describedherein. The foregoing techniques and procedures are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification. See e.g. Sambrook et al.Molecular Cloning: A Laboratory Manual (3rd ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (2001)), which isincorporated herein by reference. The nomenclatures utilized inconnection with, and the laboratory procedures and techniques of,analytical chemistry, synthetic organic chemistry, and medicinal andpharmaceutical chemistry described herein are those well known andcommonly used in the art. Standard techniques are used for chemicalsyntheses, chemical analyses, pharmaceutical preparation, formulation,and delivery, and treatment of patients.

INCORPORATION BY REFERENCE

All references cited herein, including patents, patent applications,papers, text books, and the like, including the references citedtherein, are hereby incorporated herein by reference in their entirety.

TABLE OF CDR SEQUENCES SEQ SEQ SEQ Ab ID ID ID chain Pat ID: NO: CDR1NO: CDR2 NO: CDR3 heavy U1-1 235 GGSINSGDYYWS 258 YIYYSGSTYYNPSLKS 283ADYDFWSGYFDY light U1-1 318 RASQGIRNDLG 343 AASSLQS 360 LQHNSYPWT heavyU1-2 236 GGSISSGDYYWS 259 YIYYSGSTYYNPSLRS 283 ADYDFWSGYFDY light U1-2318 RASQGIRNDLG 343 AASSLQS 361 LQHNGYPWT heavy U1-3 237 GGSISSGGYYWS258 YIYYSGSTYYNPSLKS 284 DGYDSSGYYHGYFDY light U1-3 319 KSSQSVLYSSNNK344 WASTRES 362 QQYYSTPLT NYLA heavy U1-4 236 GGSISSGDYYWS 258YIYYSGSTYYNPSLKS 283 ADYDFWSGYFDY light U1-4 318 RASQGIRNDLG 343 AASSLQS363 LQHNNYPWT heavy U1-5 236 GGSISSGDYYWS 258 YIYYSGSTYYNPSLKS 283ADYDFWSGYFDY light U1-5 318 RASQGIRNDLG 343 AASSLQS 364 LQHNTYPWT heavyU1-6 236 GGSISSGDYYWS 258 YIYYSGSTYYNPSLKS 285 ADYDFWNGYFDY light U1-6318 RASQGIRNDLG 343 AASSLQS 364 LQHNTYPWT heavy U1-7 236 GGSISSGDYYWS258 YIYYSGSTYYNPSLKS 283 ADYDFWSGYFDY light U1-7 320 RASQDIRNDLG 343AASSLQS 360 LQHNSYPWT heavy U1-8 238 GYTLTELSMY 260 GFDPEDGETIYAQKFQ 286GWNYVFDY G light U1-8 321 RSSQSLLHSNGYN 345 LDSHRAS 365 MQALQTPLT YLDheavy U1-9 236 GGSISSGDYYWS 258 YIYYSGSTYYNPSLKS 285 ADYDFWNGYFDY lightU1-9 320 RASQDIRNDLG 343 AASSLQS 360 LQHNSYPWT heavy U1-10 236GGSISSGDYYWS 258 YIYYSGSTYYNPSLKS 283 ADYDFWSGYFDY light U1-10 318RASQGIRNDLG 343 AASSLQS 363 LQHNNYPWT heavy U1-11 236 GGSISSGDYYWS 258YIYYSGSTYYNPSLKS 283 ADYDFWSGYFDY light U1-11 318 RASQGIRNDLG 343AASSLQS 364 LQHNTYPWT heavy U1-12 236 GGSISSGDYYWS 258 YIYYSGSTYYNPSLKS283 ADYDFWSGYFDY light U1-12 318 RASQGIRNDLG 343 AASSLQS 363 LQHNNYPWTheavy U1-13 237 GGSISSGGYYWS 258 YIYYSGSTYYNPSLKS 287 EDDGMDV lightU1-13 322 RSSQSLLHSNGYN 346 LGSNRAS 366 MQALQTPIT YLE heavy U1-14 236GGSISSGDYYWS 258 YIYYSGSTYYNPSLKS 283 ADYDFWSGYFDY light U1-14 318RASQGIRNDLG 343 AASSLQS 364 LQHNTYPWT heavy U1-15 239 GGSVSSGGYYWS 261YIYYSGSTNYNPSLKS 288 DGDVDTAMVDAFDI light U1-15 323 RASQSLSGNYLA 347GASSRAT 367 QQYDRSPLT heavy U1-16 236 GGSISSGDYYWS 258 YIYYSGSTYYNPSLKS283 ADYDFWSGYFDY light U1-16 318 RASQGIRNDLG 343 AASSLQS 360 LQHNSYPWTheavy U1-17 236 GGSISSGDYYWS 262 YIYYSGSTYYNSSLKS 283 ADYDFWSGYFDY lightU1-17 318 RASQGIRNDLG 343 AASSLQS 360 LQHNSYPWT heavy U1-18 236GGSISSGDYYWS 258 YIYYSGSTYYNPSLKS 283 ADYDFWSGYFDY light U1-18 318RASQGIRNDLG 343 AASSLQS 360 LQHNSYPWT heavy U1-19 236 GGSISSGDYYWS 258YIYYSGSTYYNPSLKS 289 GDYDFWSGEFDY light U1-19 sequence not availableheavy U1-20 237 GGSISSGGYYWS 263 YIYDSGSTYYNPSLKS 290 DQGQDGYSYGYGYYYGMDV light U1-20 324 QASQDISNYLN 348 VASNLET 368 QQCDNLPLT heavy U1-21236 GGSISSGDYYWS 258 YIYYSGSTYYNPSLKS 283 ADYDFWSGYFDY light U1-21 320RASQDIRNDLG 349 AASRLQS 360 LQHNSYPWT heavy U1-22 236 GGSISSGDYYWS 258YIYYSGSTYYNPSLKS 283 ADYDFWSGYFDY light U1-22 318 RASQGIRNDLG 350AASSLQN 360 LQHNSYPWT heavy U1-23 236 GGSISSGDYYWS 258 YIYYSGSTYYNPSLKS283 ADYDFWSGYFDY light U1-23 318 RASQGIRNDLG 343 AASSLQS 360 LQHNSYPWTheavy U1-24 236 GGSISSGDYYWS 258 YIYYSGSTYYNPSLKS 285 ADYDFWNGYFDY lightU1-24 318 RASQGIRNDLG 343 AASSLQS 363 LQHNNYPWT heavy U1-25 236GGSISSGDYYWS 258 YIYYSGSTYYNPSLKS 283 ADYDFWSGYFDY light U1-25 318RASQGIRNDLG 350 AASSLQN 360 LQHNSYPWT heavy U1-26 236 GGSISSGDYYWS 258YIYYSGSTYYNPSLKS 291 ADYDFWSGYFDF light U1-26 318 RASQGIRNDLG 343AASSLQS 361 LQHNGYPWT heavy U1-27 236 GGSISSGDYYWS 258 YIYYSGSTYYNPSLKS291 ADYDFWSGYFDF light U1-27 318 RASQGIRNDLG 343 AASSLQS 361 LQHNGYPWTheavy U1-28 236 GGSISSGDYYWS 258 YIYYSGSTYYNPSLKS 292 ADYDFWSGYFDS lightU1-28 318 RASQGIRNDLG 343 AASSLQS 361 LQHNGYPWT heavy U1-29 240GFTFNSYDMH 264 VIWYDGSNKYYADSVK 293 DRLCTNGVCYEDYGMD G V light U1-29 324QASQDISNYLN 351 DASNLET 369 QHYDTLPLT heavy U1-30 236 GGSISSGDYYWS 265YIYYSGTTYYNPSLKS 283 ADYDFWSGYFDY light U1-30 325 RAGQGIRNDLG 343AASSLQS 360 LQHNSYPWT heavy U1-31 241 GYTFTNYGIS 266 WISAYDGYRNYAQKLQ294 DVQDYGDYDYFDY G light U1-31 326 RASQSISSYLN 343 AASSLQS 370QQSYSTPIT heavy U1-32 236 GGSISSGDYYWS 265 YIYYSGTTYYNPSLKS 283ADYDFWSGYFDY light U1-32 325 RAGQGIRNDLG 343 AASSLQS 360 LQHNSYPWT heavyU1-33 236 GGSISSGDYYWS 258 YIYYSGSTYYNPSLKS 295 ADYDFWSGHFDC light U1-33327 RASQGIRDDLG 352 AESSLQS 371 LQHHSYPWT heavy U1-34 241 GYTFTNYGIS 266WISAYDGYRNYAQKLQ 294 DVQDYGDYDYFDY G light U1-34 326 RASQSISSYLN 343AASSLQS 370 QQSYSTPIT heavy U1-35 242 GFTFSDYYMS 267 YISSSGNNIYHADSVK296 ERYSGYDDPDGFDI G light U1-35 328 QASQDISNYLS 351 DASNLET 372QQYDNPPCS heavy U1-36 243 GGSISSGYYYWS 268 YIYYSGTTYYNPSFKS 297ADYDFWSGHFDY light U1-36 318 RASQGIRNDLG 343 AASSLQS 360 LQHNSYPWT heavyU1-37 244 GYTFTSYGIS 269 WISAYDGHTNYAQKLQ 298 DPHDYSNYEAFDF G lightU1-37 326 RASQSISSYLN 343 AASSLQS 370 QQSYSTPIT heavy U1-38 245GFSLSTSGVGVG 270 LIYWNDDKRYSPSLKS 299 RDEVRGFDY light U1-38 329RSSQSLVYSDGYT 353 KVSNWDS 373 MQGAHWPIT YLH heavy U1-39 246 GFTVSSNYMS271 VIYSGGSTYYADSVKG 300 GQWLDV light U1-39 321 RSSQSLLHSNGYN 354LGFHRAS 374 RQALQTPLT YLD heavy U1-40 237 GGSISSGGYYWS 272YIYSSGSTYYNPSLKS 301 DRELELYYYYYGMDV light U1-40 330 RSSQSLLYSNGYN 346LGSNRAS 365 MQALQTPLT YLD heavy U1-41 237 GGSISSGGYYWS 258YIYYSGSTYYNPSLKS 302 DRELEGYSNYYGVDV light U1-41 331 RASQAISNYLN 343AASSLQS 375 QQNNSLPIT heavy U1-42 247 GYSFTSYWIG 273 IIYPGDSDTRYSPSFQ303 HENYGDYNY G light U1-42 332 RASQSIRSYLN 343 AASSLQS 376 QQSNGSPLTheavy U1-43 237 GGSISSGGYYWS 259 YIYYSGSTYYNPSLRS 304 DREREWDDYGDPQGMD Vlight U1-43 333 RASQSISSYLH 343 AASSLQS 377 QQSYSNPLT heavy U1-44 247GYSFTSYWIG 274 IIWPGDSDTIYSPSFQ 303 HENYGDYNY G light U1-44 332RASQSIRSYLN 343 AASSLQS 378 QQSISSPLT heavy U1-45 248 GYTFTSYDIN 275WMNPNSGDTGYAQVFQ 305 FGDLPYDYSYYEWFDP G light U1-45 326 RASQSISSYLN 343AASSLQS 379 QQSYSTPLT heavy U1-46 249 GDSVSSNSAAWN 276 RTYYRSKWYNDYAVSV306 DLYDFWSGYPYYYGMD KS V light U1-46 sequence not available heavy U1-47249 GDSVSSNSAAWN 276 RTYYRSKWYNDYAVSV 307 DYYGSGSFYYYYGMDV KS lightU1-47 326 RASQSISSYLN 355 AASNLQS 380 QQSYSTPRT heavy U1-48 250GGSISSYYWS 277 HIYTSGSTNYNPSLKS 308 EAIFGVGPYYYYGMDV light U1-48sequence not available heavy U1-49 251 GYTFTGYYMH 278 WINPNIGGTNCAQKFQ309 GGRYSSSWSYYYYGMD G V light U1-49 334 KSSQSLLLSDGGT 356 EVSNRFS 381MQSMQLPIT YLY heavy U1-50 239 GGSVSSGGYYWS 261 YIYYSGSTNYNPSLKS 310GGDSNYEDYYYYYGMD V light U1-50 335 RASQSISIYLH 343 AASSLQS 382 QQSYTSPITheavy U1-51 250 GGSISSYYWS 261 YIYYSGSTNYNPSLKS 311 DSSYYDSSGYYLYYYA MDVlight U1-51 319 KSSQSVLYSSNNK 344 WASTRES 383 QQYYTTPLT NYLA heavy U1-52237 GGSISSGGYYWS 279 NIYYSGSTYYNPSLKS 312 GGTGTNYYYYYGMDV light U1-52336 RASQSVSSSYLA 357 GASSWAT 384 QQYGSSPLT heavy U1-53 252 GFTFSIYSMN280 YISSSSSTIYYADSVK 313 DRGDFDAFDI G light U1-53 337 QASQDITNYLN 351DASNLET 385 QQCENFPIT heavy U1-55.1 253 GGSVSSGGYYWN 281YINYSGSTNYNPSLKS 301 DRELELYYYYYGMDV light U1-55.1 will be same as U1-55heavy U1-55 will be same as U1-55.1 light U1-55 338 RSSQSLLYSNGYK 346LGSNRAS 366 MQALQTPIT YLD heavy U1-57.1 will be same as U1-57 lightU1-57.1 338 RSSQSLLYSNGYK 346 LGSNRAS 366 MQALQTPIT YLD heavy U1-57 254GGSVSSGGYYWN 281 YINYSGSTNYNPSLKS 301 DRELELYYYYYGMDV light U1-57 willbe same as U1-57.1 heavy U1-58 255 GFTFSSYGMH 264 VIWYDGSNKYYADSVK 314AARLDYYYGMDV G light U1-58 339 RASQSINSYLN 358 GASGLQS 386 QQSYSSPLTheavy U1-59 256 GGSFSGYYWS 282 EINHSGSTNYNPSLKS 315 DKWTWYFDL lightU1-59 340 RSSQSVLYSSSNR 344 WASTRES 387 QQYYSTPRT NYLA heavy U1-61.1 257GVSISSGGYYWS 258 YIYYSGSTYYNPSLKS 316 DSESEYSSSSNYGMDV light U1-61.1will be the same as U1-61.1 heavy U1-61 257 GVSISSGGYYWS 258YIYYSGSTYYNPSLKS 316 DSESEYSSSSNYGMDV light U1-61 341 RASQTISSYLN 359AASSLQG 377 QQSYSNPLT heavy U1-62 247 GYSFTSYWIG 273 IIYPGDSDTRYSPSFQ317 QMAGNYYYGMDV G light U1-62 342 RASQSVISIYLA 347 GASSRAT 388QQYGSSPCS

1. An isolated binding protein that binds to HER-3, wherein said bindingprotein comprises a heavy chain amino acid sequence that comprises aCDRH1 selected from the group consisting of SEQ ID NOs:236, 251, 252,and 256, a CDRH2 selected from the group consisting of SEQ ID NOs:258,278, 280, and 282, and a CDRH3 selected from the group consisting of SEQID NOs:283, 285, 309, 313, and 315, and a light chain amino acidsequence that comprises a CDRL1 selected from the group consisting ofSEQ ID NOs:320, 334, 337, and 340, a CDRL2 selected from the groupconsisting of SEQ ID NOs: 343, 356, 351, and 344, and a CDRL3 selectedfrom the group consisting of SEQ ID NOs:360, 381, 385, and
 387. 2. Anisolated binding protein that binds to HER3-, comprising: a heavy chainamino acid sequence selected from the group consisting of SEQ ID NOs:42, 70, 92, and
 96. 3. The isolated binding protein of claim 2comprising: a light chain amino acid sequence selected from the groupconsisting of SEQ ID NOs: 44, 56, 72, 94, and
 98. 4. An isolated bindingprotein that binds HER-3, comprising: a heavy chain amino acid sequenceselected from the group consisting of SEQ ID NOs: 42, 54, 70, 92, and96; and a light chain amino acid sequence selected from the groupconsisting of SEQ ID NOs: 44, 56, 72, 94, and
 98. 5. An isolated bindingprotein that binds to HER-3, comprising the heavy chain amino acidsequence of SEQ ID NO:42 and the light chain amino acid sequence of SEQID NO:44.
 6. An isolated binding protein that binds to HER-3, comprisingthe heavy chain amino acid sequence of SEQ ID NO:54 and the light chainamino acid sequence of SEQ ID NO:56.
 7. The isolated binding proteinthat binds to HER-3, comprising the heavy chain amino acid sequence ofSEQ ID NO:70 and the light chain amino acid sequence of SEQ ID NO:72. 8.The isolated binding protein of any of claims 1-4, or 5-7, wherein thebinding protein is directed against the extracellular domain of HER-3.9. The isolated binding protein of any one of claims 1-4, or 5-7,wherein the binding of the binding protein to HER-3 reducesHER-3-mediated signal transduction.
 10. The isolated binding protein ofany one of claims 1-4, or 5-7, wherein the binding of the bindingprotein to HER-3 reduces HER-3 phosphorylation.
 11. The isolated bindingprotein of any one of claims 1-4, or 5-7, wherein the binding of thebinding protein to HER-3 reduces cell proliferation.
 12. The isolatedbinding protein of any one of claims 1-4, or 5-7, wherein the binding ofthe binding protein to HER-3 reduces cell migration.
 13. The isolatedbinding protein of any one of claims 1-4, or 5-7, wherein the binding ofthe binding protein to HER-3 increases the downregulation of HER-3. 14.The isolated binding protein of any one of claims 1-4, or 5-7, which isan antibody.
 15. The isolated binding protein of claim 14, wherein theantibody is a monoclonal antibody, a polyclonal antibody, a recombinantantibody, a humanized antibody, a human antibody, a chimeric antibody, amultispecific antibody, or an antibody fragment thereof.
 16. Theisolated binding protein of claim 15, wherein the antibody fragment is aFab fragment, a Fab′ fragment, a F(ab′)2 fragment, a Fv fragment, adiabody, or a single chain antibody molecule.
 17. The isolated bindingprotein of claim 14, wherein said isolated binding protein is of theIgG1-, IgG2-, IgG3- or IgG4-type.
 18. The isolated binding protein ofclaim 14, wherein the binding protein is coupled to a labelling group.19. The isolated binding protein of claim 18, wherein the labellinggroup is a radioisotope or radionuclide, a fluorescent group, anenzymatic group, a chemiluminescent group, a biotinyl group, or apredetermined polypeptide epitope.
 20. The isolated binding protein ofclaim 14, wherein the binding protein is coupled to an effector group.21. The isolated binding protein of claim 20, wherein the effector groupis a radioisotope or radionuclide, a toxin, or a therapeutic orchemotherapeutic group.
 22. The isolated binding protein of claim 21,wherein the therapeutic or chemotherapeutic group is selected from thegroup consisting of calicheamicin, auristatin-PE, geldanamycin,maytansine and derivatives thereof.
 23. A pharmaceutical compositioncomprising as an active agent at least one isolated binding protein ofany one of claims 2-4, or 5-7, and a pharmaceutically acceptablecarrier, diluent or adjuvant.
 24. The composition of claim 23 fortherapeutic use.
 25. The composition of claim 23 for diagnostic use. 26.A kit comprising the isolated binding protein of any one of claims 1-4or 5-7.
 27. The kit of claim 26, comprising a further therapeutic agent.28. The kit of claim 27 wherein the further therapeutic agent is anantineoplastic agent.
 29. The kit of claim 28, wherein theanti-neoplastic agent is an anti-tumor antibody or a chemotherapeuticagent.